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Digital PCR

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

GEN Roundup: Digital PCR Advances Partition by Partition  

By Partitioning Samples Digital PCR Is Lowering Detection Limits and Enabling New Applications

GEN  Mar 1, 2016 (Vol. 36, No. 5)       http://www.genengnews.com/gen-articles/gen-roundup-digital-pcr-advances-partition-by-partition/5697

 

  • Digital PCR (dPCR) has generated intense interest because it is showing potential as a clinical diagnostics tool. It has already proven to be a useful technique for any application where extreme sensitivity or precise quantification is essential, such as identifying mutations or copy number variations in tumor cells, or examining gene expression at the single-cell level.

    GEN interviewed several dPCR experts to find out specifically why the technique is increasing in popularity. GEN also asked the experts to envision dPCR’s future capabilities.

  • GEN: What makes dPCR technology such a superior tool for discovery and diagnostic applications?

    Dr. Shelton The high levels of sensitivity, precision, and reproducibility in DNA and quantification are the major strengths of dPCR. The technology is robust where differences in primer efficiency or the presence of sample-specific PCR inhibitors are trivial to the final quantification through an end-point amplification reaction.

    This provides value to discovery as a trusted tool for validating potential biomarkers and hypotheses generated by broad profiling techniques such as microarrays or next-generation sequencing (NGS). In diagnostics applications, the reproducibility and rapid results of dPCR are critical for labs around the world to quickly compare and share data, especially for ultra-low detection of DNA where variability is high.

    Dr. Garner Digital PCR provides a precise direct counting approach for single molecule detection, thereby providing a straightforward process for the absolute quantification of nucleic acids in samples. One of the biggest advantages of using a system such as ours is its ability to do real-time reads on digital samples. When samples go through PCR, their results are recorded after each cycle.

    These results build a curve, and customers can analyze the data if something went wrong. If it isn’t a clean read—from either a contamination issue, primer-dimer issue, or off-target issue—the curve isn’t the classic PCR curve.

    Dr. Menezes Digital PCR allows absolute quantification of target concentration in samples without the need for standard curves. Obtaining consistent, precise, and absolute quantification with regular qPCR is dependent on standard curve generation and amplification efficiency calculations, which can introduce errors.

    Ms. Hibbs At MilliporeSigma Cell Design Studio, the implementation of dPCR has improved and accelerated the custom cell engineering workflow. After the application of zinc finger nuclease or CRISPR/Cas to create precise genetic modifications in mammalian cell lines, dPCR is used to characterize the expected frequency of homologous recombination and develop a screening strategy based on this expected frequency.

    In some cell lines, homologous recombination occurs at a low frequency. In such cases, dPCR is used to screen cell pools and subsequently identify rare clones having the desired mutation. Digital PCR is also used to accurately and expeditiously measure target gene copy number. It is used this way, for example, in polyploid cell lines.

    Dr. Price The ability to partition genomic samples to a level that enables robust detection of single target molecules is what sets dPCR apart as an innovative tool. Each partition (droplet in the case of the RainDrop System) operates as an individual PCR reaction, allowing for sensitive, reproducible, and precise quantification of nucleic acid molecules without the need for reference standards or endogenous controls.

    Partitioning also provides greater tolerance to PCR inhibitors compared to quantitative PCR (qPCR). In doing so, dPCR can remedy many shortcomings of qPCR by transforming the analog, exponential nature of PCR into a digital signal.

    Mr. Wakida Digital PCR is an ideal technology for detecting rare targets at concentrations of 0.1% or lower. By partitioning samples prior to PCR, exceptionally rare targets can be isolated into individual partitions and amplified.

    Digital PCR produces absolute quantitative results, so in some respects, it is easier than qPCR because it doesn’t require a standard curve, with the added advantages of being highly tolerant of inhibitors and being able to detect more minute fold changes. Absolute quantification is useful for generating reference standards, detecting viral load, and preparing NGS libraries.

  • GEN: In what field do you think dPCR will have the greatest impact in the future?

    Dr. Shelton dPCR will have a great impact on precision medicine, especially in liquid biopsy analysis. Cell-free DNA from bodily fluids such as urine or blood plasma can be analyzed quickly and cost-effectively using dPCR. For example, a rapid dPCR test can be performed to determine mutations present in a patient’s tumor and help drive treatment decisions.

    Iterative monitoring of disease states can also be achieved due to the relatively low cost of dPCR, providing faster response times when medications are failing. Gene editing will also be greatly impacted by dPCR. Digital PCR enables refinement and optimization of gene-editing tools and conditions. Digital PCR also serves as quality control of therapeutically modified cells and viral transfer vectors used in gene-therapy efforts.

    Dr. Garner The BioMark™ HD system combines dPCR with simultaneous real-time data for counting and validation. This capability is important for applications such as rare mutation detection, GMO quantitation, and aneuploidy detection—where false positives are intolerable and precision is paramount.

    Any field that requires precision and the ability to detect false positives is a likely target for Fluidigm’s dPCR. Suitable applications include detecting and quantifying cancer-causing genes in patients’ cells, viral RNA that infects bacteria, or fetal DNA in an expectant mother’s plasma.

    Dr. Menezes This technology is particularly useful for samples with low frequency sequences as, for example, those containing rare alleles, low levels of pathogen, or low levels of target gene expression. Teasing out fine differences in copy number variants is another area where this technology delivers more precise data.

    Ms. Hibbs Digital PCR overcomes limitations associated with low-abundance template material and quantification of rare mutations in a high background of wild-type DNA sequence. For this reason, dPCR is poised to have significant impacts in diverse clinical applications such as detection and quantification of rare mutations in liquid biopsies, detection of viral pathogens, and detection of copy number variation and mosaicism.

    Dr. Price Due to its high sensitivity, precision, and absolute quantification, the RainDrop dPCR has the potential to extend the range of nucleic acid analysis beyond the reach of other methods in a number of applications that could lend themselves to diagnostic, prognostic, and predictive applications. The precision of dPCR can be extremely useful in applications that require finer measures of fold change and rare variant detection.

    Digital PCR is suitable for addressing varied research and clinical challenges. These include the early detection of cancer, pathogen/viral detection and quantitation, copy number variation, rare mutation detection, fetal genetic screening, and predicting transplant rejection. Additional applications include gene expression analysis, microRNA analysis, and NGS library quantification.

    Mr. Wakida Digital PCR will have an impact on applications for detecting rare targets by enabling investigators to complement and extend their capabilities beyond traditionally employed methods. One such application is using dPCR to monitor rare targets in peripheral blood, as in liquid biopsies.

    The monitoring of peripheral blood by means of dPCR has been described in several peer-reviewed articles. In one such article, investigators considered the clinical value of Thermo’s QuantStudio™ 3D Digital PCR system for the detection of circulating DNA in metastatic colorectal cancer (Dig Liver Dis. 2015 Oct; 47(10): 884–90).

  • GEN: Is there a new technology on the horizon that will increase the speed and/or efficiency of dPCR?

    Dr. Shelton High-throughput sample analysis can be an issue with some dPCR systems. However, Bio-Rad’s Automated Droplet Generator allows labs to process 96 samples simultaneously, a capability that eliminates user-to-user variability and minimizes hands-on time.

    We also want users to get the most information from one sample. Therefore, we are focused on expanding the multiplexing capabilities of our system. In development at Bio-Rad are new technologies that increase the multiplexing capabilities without loss of specificity or accuracy in the downstream workflow.

    Dr. Garner Much of the industry direction seems to be in offering ever-higher resolution, or the ability to run more samples at the same resolution. Thus far, however, customers haven’t found commercial uses for these tools. Also, with increasing resolution and the search for even rarer mutations, the challenge of detecting false positives becomes an even bigger issue.

    Dr. Menezes Use of ZEN™ Double-Quenched Probes by IDT in digital PCR provides increased sensitivity and a lower limit of detection. Due to the second quencher, ZEN probes provide even lower background than traditional single-quenched probes. And this lower background enables increased sensitivity when analyzing samples with low copy number targets, where every droplet matters.

    Ms. Hibbs Quantification relies upon counting the number of positive partitions at the end point of the reaction. Accordingly, precision and resolution can be increased by increasing the number of partitions. We are now capable of analyzing on the order of millions of partitions per run, further extending the lower limit of detection. Additionally, the workflow is amenable to the integration of automation in order to increase throughput and standardize reaction set up.

    Dr. Price Although dPCR is still an emerging technology, there is tremendous interest in its potential clinical diagnostics applications. Enabling adoption of dPCR in the clinical lab requires addressing current gaps in workflow, cost, throughput, and turnaround time.

    Digital PCR technology has the potential for being improved significantly in two dimensions. First, one can address the problem of serially detecting positive versus negative partitions by leveraging lower-cost imaging detection technologies. Alternatively, one may capitalize on the small partition volumes to dramatically reduce the time to perform PCR. Ideally, the future will bring both capabilities to bear.

    Mr. Wakida Compared to qPCR, dPCR currently requires more hands-on time to set up experiments. We are investigating methods to address this.

 

PCR Shows Off Its Clinical Chops   

Thanks to Advances in Genomics, PCR Is Becoming More Common in Clinical Applications

  • Last May, Roche Molecular Systems announced that its cobas Liat Strep A assay received a CLIA waiver. This clinic-ready assay can detect Streptococcus pyogenes (group A ß-hemolytic streptococcus) DNA in throat swabs by targeting a segment of the S. pyogenes genome.

    Since its invention by Kary B. Mullis in 1985, the polymerase chain reaction (PCR) has become well established, even routine, in research laboratories. And now PCR is becoming more common in clinical applications, thanks to advances in genomics and the evolution of more sensitive quantitative PCR methodologies.

    Examples of clinical applications of PCR include point-of-care (POC) molecular tests for bacterial and viral detection, as well as mutation detection in liquid or tumor biopsies for patient stratification and treatment monitoring.
    Industry leaders recently participated in a CHI conference that was held in San Francisco. This conference—PCR for Molecular Medicine—encompassed research and clinical perspectives and emphasized advanced techniques and tools for effective disease diagnosis.
    To kick off the event, speakers shared their views on POC molecular tests. These tests, the speakers insisted, can provide significant value to healthcare only if they support timely decision making.
    Clinic-ready PCR platforms need to combine speed, ease of use, and accuracy. One such platform, the cobas Liat (“laboratory in a tube”), is manufactured by Roche Molecular Systems. The system employs nucleic acid purification and state-of-art PCR-based assay chemistry to enable POC sites to rapidly provide lab-quality results.
    The cobas Liat Strep A Assay detects Streptococcus pyogenes (group A β-hemolytic streptococcus) DNA by targeting a segment of the S. pyogenes genome. The operator transfers an aliquot of a throat swab sample in Amies medium into a cobas Liat Strep A Assay tube, scans the relevant tube and sample identification barcodes, and then inserts the tube into the analyzer for automated processing and result interpretation. No other operator intervention or interpretation is required. Results are ready in approximately 15 minutes.

    According to Shuqi Chen, Ph.D., vp of Point-of-Care R&D at Roche Molecular Systems, clinical studies of the cobas Liat Strep A Assay demonstrated 97.7% sensitivity when the test was used at CLIA-waived, intended-use sites, such as physicians’ offices. In comparison, rapid antigen tests and diagnostic culture have sensitivities of 70% and 81%, respectively (according to a 2009 study Tanz et al. in Pediatrics).

    The cobas Liat assay preserved the same ease-of-use and rapid turnaround as the rapid antigen tests. It addition, it provided significantly faster turnaround than the lab-based culture test, which can take 24–48 hours.

    A CLIA waiver was announced for the cobas Liat Strep A assay in May 2015. CLIA wavers have been submitted for cobas Liat flu assays, and Roche intends to extend the assay menu.

    POC tests are also moving into field applications. Coyote Bioscience has developed a novel method for one-step gene testing without nucleic acid extraction that can be as fast as 10 minutes from blood sample to result. Their portable devices for molecular diagnostics can be used as genetic biosensors to bring complex clinical testing directly to the patient.

    “Instead of sequential steps, reactions happen in parallel, significantly reducing analysis time. Buffer, enzyme, and temperature profiles are optimized to maximize sensitivity,” explained Sabrina Li, CEO, Coyote Bioscience. “Both RNA and DNA can be analyzed simultaneously from a drop of blood in the same reaction.”

    The first-generation Mini-8 system was used for Ebola detection in Africa where close to 600 samples were tested with 98.8% sensitivity. Recently in China, the Mini-8 system was applied in hospitals and small community clinics for hepatitis B and C and Bunia virus detection. The second-generation InstantGene system is currently being tested internally with clinical samples.

  • Digital PCR

    Conventional real-time PCR technology, while suited to the analysis of high-quality clinical samples, may effectively conceal amplification efficiency changes when sample quality is inconsistent. A more effective alternative, Bio-Rad suggests, is its droplet-digital PCR (ddPCR) technology, which can provide absolute quantification of target DNA or RNA, a critical advantage when samples are limited, degraded, or contain PCR inhibitors. The company says that of the half-dozen clinical trials that are using digital PCR, half rely on the Bio-Rad QX200 ddPCR system.

    Personalized cancer care requires ultra-sensitive detection and monitoring of actionable mutations from patient samples. The high sensitivity and precision of droplet-digital PCR (ddPCR) from Bio-Rad Laboratories offers critical advantages when clinical samples are limited, degraded, or contain PCR inhibitors.

    Typically, formalin-fixed and paraffin-embedded (FFPE) tissue samples are processed. FFPE samples work well for immunohistochemistry and protein analysis; however, the formalin fixation can damage nucleic acids and inhibit the PCR reaction. Samples may yield 100 ng of purified nucleic acid, but the actual amplifiable material is less than 1%, or 1 ng, in most cases.

    “Current qPCR technology depends on real-time fluorescence accumulation as the PCR is occurring, which can be an effective means of detecting and quantifying DNA targets in nondegraded samples,” commented Dawne Shelton, Ph.D., staff scientist, Digital Biology Center, Applications Development Group, Bio-Rad Laboratories. “Amplification efficiency is critical; if that amplification efficiency changes because of sample quality it is hidden in the qPCR methodology.”

    “In ddPCR, that is a big red flag,” Dr. Shelton continued. “It changes the format of how the data look immediately so you know the amount of inhibition and which samples are too inhibited to use.”

    Tissue types vary and contain different degrees of fat or other content that can also act as PCR inhibitors. In blood monitoring, the small circulating fragments of DNA are extremely degraded; in addition, food, supplements, or other compounds ingested by the patient may have an inhibitory effect.

    Clinical labs test for these variabilities and clean the blood, but remnant PCR inhibitors can remain. In ddPCR, a single template is partitioned into a droplet. If the droplet contains a good template, it produces a signal; otherwise, it does not—a simple yes or no answer.

    “Even if there is no PCR inhibition, most clinical samples yield very small amounts of nucleic acid,” Dr. Shelton added. “To make a secure decision using qPCR is difficult because you are in a gray zone at the very end of its linear range. ddPCR operates best with small sample amounts and provides good statistics for confidence in your results.”

    Currently, at least a half dozen clinical trials worldwide are using digital PCR, half of them are using the Bio-Rad QX200 Droplet Digital PCR system. Examples of studies include examining BCR-ABL monitoring in patients with chronic myelogenous leukemia (CML); identifying activating mutations in epidermal growth factor receptor (EGFR) for first-line therapy of new drugs in patients with lung cancer; and the monitoring of resistance mutations such as EFGR T790M in patients with non-small cell lung cancer (NSCLC).

    Clovis Oncology used a technology called BEAMing (Beads, Emulsions, Amplification, and Magnetics), a type of digital PCR for blood-based molecular testing, to perform EGFR testing on almost 250 patients in clinical trials. In BEAMing, individual EGFR gene copies from plasma are separated into individual water droplets in a water-in-oil emulsion. The gene copies are then amplified by PCR on magnetic beads.

    The beads are counted by flow cytometry using fluorescently labeled probes to distinguish mutant beads from wild-type. Because each bead can be traced to an individual EGFR molecule in the patient’s plasma, the method is highly quantitative.

    “BEAMing is particularly well-suited for the detection of known mutations in circulating tumor DNA. In this circumstance, the mutation of interest often occurs at low levels, perhaps only 1–2 copies per milliliter or even less, and in a high background of wild-type DNA that comes from normal tissue. BEAMing can detect one mutant molecule in a background of 5,000 wild-type molecules in clinical samples,” stated Andrew Allen, MRCP, Ph.D., chief medical officer, Clovis Oncology.

    In the studies, the EGFR-resistance mutation T790M could be identified in plasma 81% of the time that it was seen in the matched patient tumor biopsy. Additionally, about 10% of patients in the study had a T790M mutation in plasma that was not identified in tissue, presumably because of tumor heterogeneity. Another 5–10% of the patients did not provide an EGFR result, usually because the tissue biopsy had no tumor cells.

    In aggregate, these results suggest that plasma EGFR testing can be a valuable complement to tumor testing in the clinical management of NSCLC patients, and can provide an alternative when a biopsy is not available. Tumor biopsies may provide only limited tissue, if in fact any tissue is available, for molecular analysis. Also, mutations may be missed due to tumor heterogeneity. These mutations may be captured by sampling the blood, which acts as a reservoir for mutations from all parts of a patient’s tumor burden.

    In the last few years, a panoply of clinically actionable driver mutations have been identified for NSCLC, including mutations in EGFR, BRAF, and HER2, as well as ALK, ROS, and RET rearrangements. These driver mutations will migrate NSCLC molecular diagnostic testing in the next few years toward panel testing of relevant cancer genes using various digital technologies, including next-generation sequencing.

     

PCR Has a History of Amplifying Its Game

A GEN 35th Anniversary Retrospective

PCR Has a History of Amplifying Its Game

PCR is a fast and inexpensive technique used to amplify segments of DNA that continues to adapt and evolve for the demanding needs of molecular biology researchers. This diagram shows the basic principles of PCR amplification. [NHGRI]

  • The influence that the polymerase chain reaction (PCR) has had on modern molecular biology is nothing short of remarkable. This technique, which is akin to molecular photocopying, has been the centerpiece of everything from the OJ Simpson Trial to the completion of the Human Genome Project. Clinical laboratories use this DNA amplification method for infectious disease testing and tissue typing in organ transplantation. Most recently, with the explosion of the molecular diagnostics field and meteoric rise in the use of next-generation sequencing platforms, PCR has enhanced its standing as an essential pillar of genomic science.

    Let’s open the door to the past and take a look back around 35 years ago when GEN started reporting on the relatively new disciplines of genetic engineering and molecular biology. At that time, GEN was among the first to hear the buzz surrounding a new method to synthesize and amplify DNA in the laboratory. In reviewing the fascinating history of PCR, we will see how the molecular diagnostics field took shape and where it could be headed in the future.

  • Some Like It Hot

    The biological sciences rarely advance within a vacuum—rather they rely on previous discoveries to promote directly or indirectly our understanding. The contributions made by scientists in the field of molecular biology that contributed to the functional pieces of PCR were numerous and spread out over more than two decades.

    It began with H. Gobind Khorana’s advances in understanding the genetic code, leading to the use of synthetic DNA oligonucleotides, continued through Kjell Kleepe’s 1971 vision of a two-primer system for replicating DNA segments, to Fredrick Sanger’s method of DNA sequencing—a process that would win him the Nobel prize in 1980—which utilized DNA oligo primers, nucleotide precursors, and a DNA synthesis enzyme.

    All of the previous discoveries were essential to PCR’s birth, yet it would be an egregious mistake to begin a retrospective on PCR and not discuss the enzyme upon which the entire reaction hinges upon—DNA polymerase. In 1956, Nobel laureate Arthur Kornberg and his colleagues discovered DNA polymerase (Pol I), in Escherichia coli. Moreover, the researchers described the fundamental process by which the polymerase enzyme copies the base sequence of a DNA template strand. However, it would take biologists another 20 years to discover a version of DNA polymerase that was stable enough for use for any meaningful laboratory purposes.

    That discovery came in 1976 when a team of researchers from the University of Cincinnati described the activity of a DNA polymerase (Taq) they isolated from the extreme thermophile bacteria, Thermus aquaticus, which lives in hot springs and hydrothermal vents. The fact that this enzyme could withstand typical protein-denaturing temperatures and function optimally around 75–80°C fortuitously set the stage for the development of PCR.

    By 1983, all of the ingredients to bake the molecular cake were sitting in the biological cupboard waiting to be assembled in the proper order. At that time, Nobel laureate Kary Mullis was working as a scientist for the Cetus Corporation trying to perfect oligonucleotide synthesis. Mullis stumbled upon the idea of amplifying segments of DNA using multiple rounds of replication and the two primer system—essentially modifying and expanding upon Sanger’s sequencing reaction. Mullis discovered that the temperatures for each step (melting, annealing, and extension) in the reaction would need to be painstakingly controlled by hand. In addition, he realized that since the reactions were using a non-thermostable DNA polymerase, fresh enzyme would need to be “spiked in” after each successive cycle.

    Mullis’ hard work and persistence paid off as the reaction was successful at amplifying a particular segment of DNA that was flanked by two opposing nucleotide primer molecules. Two years later, the Cetus team presented their work at the annual meeting of the American Society for Human Genetics, and the first mention of the method was published in Science that same year; however, that article did not go into detail about the specifics of the newly developed PCR method—a paper that would be rejected by roughly 15 journals and would not be published until 1987.

    Although scientists were a bit slow on the uptake for the new method, the researchers at Cetus were developing ways to improve upon the original assay. In 1986, the scientists substituted the original heat-liable DNA polymerase for the temperature-resistant Taq polymerase, removing the need to spike in enzyme and dramatically reducing errors while increasing sensitivity. A year later, PerkinElmer launched their creation of a thermal cycler, allowing scientists to regulate the heating and cooling parts of the PCR reaction with greater efficiency.

    Extremely soon after the introduction of Taq and the launch of the thermal cycler, the PCR reaction exploded exponentially among research laboratories and not only vaulted molecular biology to the pinnacle of researcher interests, it also launched a molecular diagnostics revolution that continues today and shows no signs of slowing down.

  • Molecular Workhorse

    In the years since PCR first burst onto the scene, there have been a number of significant advancements to the technique that have widely improved the overall method. For example, in 1991, a new DNA polymerase from the hyperthermophilic bacteria Pyrococcus furiosus, or Pfu, was introduced as a high-fidelity alternative enzyme to Taq. Unlike Taq polymerase, Pfu has built in 3′ to 5′ exonuclease proofreading activity, which allows the enzyme to correct nucleotide incorporation errors on the fly—dramatically increasing base specificity, albeit at a reduced rate of amplification versusTaq.

    In 1995, two advancements were introduced to PCR users. The first, called antibody “hot-start” PCR, utilized an immunoglobulin molecule that is directed to the DNA polymerase and inhibits its activity until the first 95°C melt stage, denaturing the antibody and allowing the polymerase to become active. Although this process was effective in increasing the specificity of the PCR reaction, many researchers found that the technique was time consuming and often caused cross-contamination of samples.

    The second innovation introduced that year began another revolution for molecular biology and the PCR method. Real-time PCR, or quantitative PCR (qPCR), allowed researchers to quantitatively create DNA templates for PCR amplification from RNA transcripts through the use of the reverse-transcriptase enzyme and specifically incorporated fluorescent reporter dyes. The technique is still widely used by researchers to monitor gene expression extremely accurately. Over the past 20 years, many companies have spent many R&D dollars to create more accurate, higher throughput, and simple qPCR machines to meet researcher demands.

    With the advent of next-generation sequencing techniques—and the rise of techniques that started commanding the attention of more and more researchers—PCR machines and methods needed to evolve and modernize to keep pace. PCR remained the lynchpin in almost all the next-generation sequencing reactions that came along, but the traditional technique wasn’t nearly as precise as required.

    Digital PCR (dPCR) was introduced as a refinement of the conventional method, with the first real commercial system emerging around 2006. dPCR can be used to quantify directly and clonally amplify DNA or RNA.

    The apparatus carries out a single reaction within a sample. The sample, however, is separated into a large number of partitions. Moreover, the reaction is performed in each partition individually—allowing a more reliable measurement of nucleic acid content. Researchers often use this method for studying gene-sequence variations, such as copy number variants (CNV), point mutations, rare-sequence detection, and microRNA analysis, as well as for routine amplification of next-generation sequencing samples.

  • Future of PCR: Better, Faster, Stronger!

    It is almost impossible to envision a future laboratory setting that wouldn’t utilize PCR in some fashion, especially due to the heavy reliance of next-generation sequencing techniques for accurate PCR samples and at the very least using the method as a simple amplification tool for creating DNA fragments of interest.

    Yet there is at least one new next-generation sequencing technique that can identify native DNA sequences without an amplification step—nanopore sequencing. Although this technique has performed well in many preliminary trials, it is in its relative infancy. It will probably undergo additional development lasting several years before it approaches large-scale adoption by researchers. Even then, PCR has become so engrained into daily laboratory life that to try to phase out the technique would be like asking molecular biologists to give up their pipettes or restriction enzymes.

    Most PCR equipment manufacturers continue to seek ways to improve the speed and sensitivity of their thermal cyclers, while biologists continue to look toward ways to genetically engineer better DNA polymerase molecules with even greater fidelity than their naturally occurring cousins. Whatever the new advancements are, and wherever they lead the life sciences field, you can count on us at GEN to continue to provide our readers with detailed information for another 35 years … at least!

     

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New PCR Based Test May be Able to Detect Low Levels of Persistent CML, Guiding TKI therapy Choices

from CancerNetwork.com: Novel Assay Could Help Guide Treatment Cessation Decisions in CML

Reporter: Stephen J. Williams, Ph.D.

News | February 15, 2016 | Chronic Myeloid Leukemia, Hematologic Malignancies, Leukemia & Lymphoma
By Dave Levitan
A new personalized DNA-based assay can detect very low levels of persistent disease in chronic myeloid leukemia (CML) patients thought to be in deep remission, according to a new study. The test could help with treatment choices regarding cessation of tyrosine kinase inhibitor (TKI) therapy in these patients.
A number of recent studies have examined the possibility that some patients could stop TKI therapy after achieving deep molecular remission. “However, the safe introduction of a TKI-withdrawal strategy would require a reliable and cost-effective method for the identification of those patients with the lowest likelihood of relapse,” wrote study authors led by Alistair G. Reid, BSc, PhD, of Imperial College London.

Because the likelihood of relapse after withdrawal from therapy is probably related to persistence of residual disease, testing for low levels of BCR-ABL1–positive disease is key. In the new study, the researchers tested an assay using personalized DNA-based polymerase chain reaction (dPCR) involving identification of t(9;22) fusion junctions. The results were published in the Journal of Molecular Diagnostics.

They successfully mapped genomic breakpoints in 32 of 32 samples from CML patients with early-stage disease. Next, they tested 46 samples from 6 patients following treatment with a TKI and compared results to other quantitative PCR methods; 10 of the samples were used as positive controls, while the others were considered to be in deep molecular remission.

Of those 36 samples, dPCR detected persistent disease in 81%. This was more sensitive than two other PCR-based approaches, including RT-dPCR (25%) and DNA-based quantitative PCR (19%).

“The technologies described allow for the assignment of absolute quantities to both BCR-ABL1 DNA and RNA targets, facilitating for the first time direct comparison of mean expression vs cellular disease burden,” the authors wrote. They added that it remains to be explored whether the risk of relapse after withdrawal of therapy is related to just the number of CML cells or also to the degree of transcriptional activity in those cells.

“If validated in clinical trials of stopping TKIs, this technique will permit a more personalized approach to recommendations for dose reduction or drug cessation in individual patients, ensuring that therapy is withdrawn only from patients with the highest chance of long-term remission,” said study author Jane F. Apperley, MD, PhD, also of Imperial College London, in a press release. “The technique we describe, with which we successfully mapped a disease-specific junction in all patients tested, is relatively simple, cost-effective, and suited to a high-throughput laboratory.”

– See more at: http://www.cancernetwork.com/chronic-myeloid-leukemia/novel-assay-could-help-guide-treatment-cessation-decisions-cml?GUID=D63BFB74-A7FD-4892-846F-A7D1FFE0F131&XGUID=&rememberme=1&ts=09032016#sthash.EAzX9VUl.dpuf

– See more at: http://www.cancernetwork.com/chronic-myeloid-leukemia/novel-assay-could-help-guide-treatment-cessation-decisions-cml?GUID=D63BFB74-A7FD-4892-846F-A7D1FFE0F131&XGUID=&rememberme=1&ts=09032016#sthash.EAzX9VUl.dpuf

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A Primer on DNA and DNA Replication

Reporter and Curator: Larry H. Bernstein, MD, FCAP 

 

 

This is the FIRST discussion of a several part series leading from the genome, to protein synthesis (1), posttranslational modification of proteins (2), examples of protein effects on metabolism and signaling pathways (3), and leading to disruption of signaling pathways in disease (4), and effects leading to mutagenesis.

1.  A Primer on DNAand DNA Replication

 

dna-replication-primer-synthesis

 

dna-replication-unwinding

 

dna-replication-ligation

 

dna-replication-primer-removal

 

dna-replication-leading-strand

 

dna-replication-lagging-strand

 

dna-replication-termination

 

 

Polymerase Chain Reaction

Polymerase Chain Reaction

 

 

 

 

 

2. Overview of translational medicine

3. Genes, proteomes, and their interaction

4. Regulation of somatic stem cell Function

5.  Proteomics – The Pathway to Understanding and Decision-making in Medicine

6.  Genomics, Proteomics and standards

7.  Long Non-coding RNAs Can Encode Proteins After All

8.  Proteins and cellular adaptation to stress

9.  Loss of normal growth regulation

 

 

A Primer on DNA and DNA Replication

 

DNA Replication

DNA carries the information for making all of the cell’s proteins. These pro­teins implement all of the functions of a living organism and determine the organism’­s characteristics. When the cell reproduces, it has to pass all of this information on to the daughter cells.

Before a cell can reproduce, it must first replicate, or make a copy of, its DNA. Where DNA replication occurs depends upon whether the cells is a prokaryote or a eukaryote (see the RNA sidebar on the previous page for more about the types of cells). DNA replication occurs in the cytoplasm of prokaryotes and in the nucleus of eukaryotes. Regardless of where DNA replication occurs, the basic process is the same.

The structure of DNA lends itself easily to DNA replication. Each side of the double helix runs in opposite (anti-parallel) directions. The beauty of this structure is that it can unzip down the middle and each side can serve as a pattern or template for the other side (called semi-conservative replication). However, DNA does not unzip entirely. It unzips in a small area called a replication fork, which then moves down the entire length of the molecule.

Eukaryotic DNA replication (Wikipedia), is a conserved mechanism that restricts DNA replication to only once per cell cycle. Eukaryotic DNA replication of chromosomal DNA is central for the duplication of a cell and is necessary for the maintenance of the eukaryotic genome.

DNA replication is the action of DNA polymerases synthesizing a DNA strand complementary to the original template strand. To synthesize DNA, the double-stranded DNA is unwound by DNA helicases ahead of polymerases, forming a replication fork containing two single-stranded templates.

Replication processes permit the copying of a single DNA double helix into two DNA helices, which are divided into the daughter cells at mitosis. The major enzymatic functions carried out at the replication fork are well conserved from prokaryotes to eukaryotes, but the replication machinery in eukaryotic DNA replication is a much larger complex, coordinating many proteins at the site of replication, forming the replisome.[1]

The replisome is responsible for copying the entirety of genomic DNA in each proliferative cell. This process allows for the high-fidelity passage of hereditary/genetic information from parental cell to daughter cell and is thus essential to all organisms. Much of the cell cycle is built around ensuring that DNA replication occurs without errors.[1]

In G1 phase of the cell cycle, many of the DNA replication regulatory processes are initiated. In eukaryotes, the vast majority of DNA synthesis occurs during S phase of the cell cycle, and the entire genome must be unwound and duplicated to form two daughter copies. During G2, any damaged DNA or replication errors are corrected. Finally, one copy of the genomes is segregated to each daughter cell at mitosis or M phase.[2] These daughter copies each contain one strand from the parental duplex DNA and one nascent antiparallel strand.

This mechanism is conserved from prokaryotes to eukaryotes and is known as semiconservative DNA replication. The process of semiconservative replication for the site of DNA replication is a fork-like DNA structure, the replication fork, where the DNA helix is open, or unwound, exposing unpaired DNA nucleotides for recognition and base pairing for the incorporation of free nucleotides into double-stranded DNA.[3]

 

Let’s look at the details:

  1. An enzyme called DNA gyrase makes a nick in the double helix and each side separates
  2. An enzyme called helicase unwinds the double-stranded DNA
  3. Several small proteins called single strand binding proteins(SSB) temporarily bind to each side and keep them separated
  4. An enzyme complex called DNA polymerase“walks” down the DNA strands and adds new nucleotides to each strand. The nucleotides pair with the complementary nucleotides on the existing stand (A with T, G with C).
  5. A subunit of the DNA polymerase proofreads the new DNA
  6. An enzyme called DNA ligaseseals up the fragments into one long continuous strand
  7. The new copies automatically wind up again

Different types of cells replicated their DNA at different rates. Some cells constantly divide, like those in your hair and fingernails and bone marrow cells. Other cells go through several rounds of cell division and stop (including specialized cells, like those in your brainmuscle and heart). Finally, some cells stop dividing, but can be induced to divide to repair injury (such as skin cells and liver cells). In cells that do not constantly divide, the cues for DNA replication/cell division come in the form of chemicals. These chemicals can come from other parts of the body (hormones) or from the environment.

 

 

Pre-replicative_complex

Pre-replicative_complex

 

 

 

 

Diagram of the formation of the pre-replicative complex transforming into an active replisomeMcm 2-7 complex loads onto DNA at replication origins during G1 and unwinds DNA ahead of replicative polymerases.Cdc6 and Cdt1 bring Mcm complexes to replication origins. CDK/DDK-dependent phosphorylation of pre-replicative proteins leads toreplisome assembly and origin firing. Cdc6 and Cdt1 are no longer required and are removed from the nucleus or degraded. Mcms and associated proteins, GINS and Cdc45, unwind DNA to expose template DNA. At this point replisome assembly is completed and replication is initiated. “P” represents phosphorylation.

 

Minichromosome Maintenance Protein Complex[edit]

Main article: Minichromosome maintenance

The assembly of the minichromosome maintenance (Mcm) proteins function together as a complex in the cell. The assembly of the Mcm proteins onto chromatin requires the coordinated function of the Origin Recognition Complex (ORC), Cdc6, and Cdt1.[18] Once the Mcm proteins have been loaded onto the chromatin, ORC and Cdc6 can be removed from the chromatin without preventing subsequent DNA replication. This suggests that the primary role of the pre-replication complex is to correctly load the Mcm proteins.[19]

The Mcm proteins support roles both in the initiation and elongation steps of DNA synthesis.[20] Each Mcm protein is highly related to all others, but unique sequences distinguishing each of the subunit types are conserved across eukaryotes. All eukaryotes have exactly six Mcm protein analogs that each fall into one of the existing classes (Mcm2-7), which suggests that each Mcm protein has a unique and important function.[21]

Minichromosome maintenance proteins have been found to be required for DNA helicase activity and inactivation of any of the six Mcm proteins prevents further progression of the replication fork. This is consistent with the requirement of ORC, Cdc6, and Cdt1 function to assemble the Mcm proteins at the origin of replication.[22] The complex containing all six Mcm proteins creates a hexameric, doughnut like structure with a central cavity.[23] The helicase activity of the Mcm protein complex raises the question of how the ring-like complex is loaded onto the single-stranded DNA. One possibility is that the helicase activity of the Mcm protein complex can oscillate between an open and a closed ring formation to allow single-stranded DNA loading.[6]

Along with the minichromosome maintenance protein complex helicase activity, the complex also has associated ATPase activity.[24] A mutation in any one of the six Mcm proteins reduces the conserved ATP binding sites, which indicates that ATP hydrolysis is a coordinated event involving all six subunits of the Mcm complex.[25] Studies have shown that within the Mcm protein complex are specific catalytic pairs of Mcm proteins that function together to coordinate ATP hydrolysis. For example, Mcm3 but not Mcm6 can activate Mcm6 activity. These studies suggest that the structure for the Mcm complex is a hexamer with Mcm3 next to Mcm7Mcm2 next to Mcm6, and Mcm4 next to Mcm5. Both members of the catalytic pair contribute to the conformation that allows ATP binding and hydrolysis and the mixture of active and inactive subunits create a coordinated ATPase activity that allows the Mcm protein complex to complete ATP binding and hydrolysis as a whole.[26]

The nuclear localization of the minichromosome maintenance proteins is regulated in budding yeast cells. The Mcm proteins are present in the nucleus in G1 stage and S phase of the cell cycle, but are exported to the cytoplasm during the G2 stage and M phase. A complete and intact six subunit Mcm complex is required to enter into the cell nucleus.[27] InS. cerevisiaenuclear export is promoted by cyclin-dependent kinase (CDK) activity. Mcm proteins that are associated with chromatin are protected from CDK export machinery due to the lack of accessibility to CDK.[28]

 

Initiation Complex[edit]

During the G1 stage of the cell cycle, the replication initiation factors, origin recognition complex (ORC), Cdc6, Cdt1, and minichromosome maintenance (Mcm) protein complex, bind sequentially to DNA to form the pre-replication complex (pre-RC). At the transition of the G1 stage to the S phase of the cell cycle, S phase–specific cyclin-dependent protein kinase (CDK) and Cdc7/Dbf4 kinase (DDK) transform the pre-RC into an active replication fork. During this transformation, the pre-RC is disassembled with the loss of Cdc6, creating the initiation complex. In addition to the binding of the Mcm proteins, cell division cycle 45 (Cdc45) protein is also essential for initiating DNA replication.[29][30] Studies have shown that Mcm is critical for the loading of Cdc45 onto chromatin and this complex containing both Mcm and Cdc45 is formed at the onset of the S phase of the cell cycle.[31][32] Cdc45 targets the Mcm protein complex, which has been loaded onto the chromatin, as a component of the pre-RC at the origin of replication during the G1 stage of the cell cycle.[20]

GINS[edit]

The six minichromosome maintenance proteins and Cdc45 are essential during initiation and elongation for the movement of replication forks and for unwinding of the DNA. GINS are essential for the interaction of Mcm and Cdc45 at the origins of replication during initiation and then at DNA replication forks as the replisome progresses.[37][38] The GINS complex is composed of four small proteins Sld5 (Cdc105), Psf1 (Cdc101), Psf2 (Cdc102) and Psf3 (Cdc103), GINS represents ‘go, ichi, ni, san’ which means ‘5, 1, 2, 3’ in Japanese.[39]

Mcm10[edit]

Main article: MCM10

Mcm10 is essential for chromosome replication and interacts with the minichromosome maintenance 2-7 helicase that is loaded in an inactive form at origins of DNA replication. Mcm10 chaperones the catalytic DNA polymerase α and helps stabilize the polymerase.[40]

DDK and CDK Kinases[edit]

Main article: Cyclin-dependent kinase

At the onset of S phase, the pre-replicative complex must be activated by two S phase-specific kinases in order to form an initiation complex at an origin of replication. One kinase is the Cdc7-Dbf4 kinase called Dbf4-dependent kinase (DDK) and the other is cyclin-dependent kinase (CDK).[41] Chromatin-binding assays of Cdc45 in yeast and Xenopus have shown that a downstream event of CDK action is loading of Cdc45 onto chromatin.[30][31] Cdc6 has been speculated to be a target of CDK action, because of the association between Cdc6 and CDK, and the CDK-dependent phosphorylation of Cdc6. The CDK-dependent phosphorylation of Cdc6 has been considered to be required for entry into the S phase.[42]

Elongation[edit]

Eukaryotic_replisome_complex

 

 

 

 

Eukaryotic replisome complex and associated proteins.

The formation of the pre-replicative complex (pre-RC) marks the potential sites for the initiation of DNA replication. Consistent with the minichromosome maintenance complex encircling double stranded DNA, formation of the pre-RC does not lead to the immediate unwinding of origin DNA or the recruitment of DNA polymerases. Instead, the pre-RC that is formed during the G1 of the cell cycle is only activated to unwind the DNA and initiate replication after the cells pass from the G1 to the S phase of the cell cycle.[2]

Once the initiation complex is formed and the cells pass into the S phase, the complex then becomes a replisome. The eukaryotic replisome complex is responsible for coordinating DNA replication. Replication on the leading and lagging strands is performed by DNA polymerase ε and DNA polymerase δ. Many replisome factors including Claspin, And1, replication factor C clamp loader and the fork protection complex are responsible for regulating polymerase functions and coordinating DNA synthesis with the unwinding of the template strand by Cdc45-Mcm-GINS complex. As the DNA is unwound the twist number decreases. To compensate for this the writhe number increases, introducing positive supercoils in the DNA. These supercoils would cause DNA replication to halt if they were not removed. Topoisomerases are responsible for removing these supercoils ahead of the replication fork.

Replication Fork[edit]

The replication fork is the junction the between the newly separated template strands, known as the leading and lagging strands, and the double stranded DNA. Since duplex DNA is antiparallel, DNA replication occurs in opposite directions between the two new strands at the replication fork, but all DNA polymerases synthesize DNA in the 5′ to 3′ direction with respect to the newly synthesized strand. Further coordination is required during DNA replication. Two replicative polymerases synthesize DNA in opposite orientations. Polymerase ε synthesizes DNA on the “leading” DNA strand continuously as it is pointing in the same direction as DNA unwinding by the replisome. In contrast, polymerase δ synthesizes DNA on the “lagging” strand, which is the opposite DNA template strand, in a fragmented or discontinuous manner.

The discontinuous stretches of DNA replication products on the lagging strand are known as Okazaki fragments and are about 100 to 200 bases in length at eukaryotic replication forks. The lagging strand usually contains longer stretches of single-stranded DNA that is coated with single-stranded binding proteins, which help stabilize the single-stranded templates by preventing a secondary structure formation. In eukaryotes, these single-stranded binding proteins are a heterotrimeric complex known as replication protein A(RPA).[56]

Each Okazaki fragment is preceded by an RNA primer, which is displaced by the procession of the next Okazaki fragment during synthesis. RNAse H recognizes the DNA:RNA hybrids that are created by the use of RNA primers and is responsible for removing these from the replicated strand, leaving behind a primer:template junction. DNA polymerase α, recognizes these sites and elongates the breaks left by primer removal. In eukaryotic cells,

Replication_fork.svg

 

 

Depiction of DNA replication at replication fork. a: template strands, b: leading strand, c: lagging strand, d: replication fork, e: RNA primer, f: Okazaki fragment

Leading Strand

Lagging Strand

Replicative DNA Polymerases

 

After the replicative helicase has unwound the parental DNA duplex, exposing two single-stranded DNA templates, replicative polymerases are needed to generate two copies of the parental genome. DNA polymerase function is highly specialized and accomplish replication on specific templates and in narrow localizations. At the eukaryotic replication fork, there are three distinct replicative polymerase complexes that contribute to DNA replication: Polymerase α, Polymerase δ, and Polymerase ε. These three polymerases are essential for viability of the cell.[66]

Because DNA polymerases require a primer on which to begin DNA synthesis, polymerase α (Pol α) acts as a replicative primase. Pol α is associated with an RNA primase and this complex accomplishes the priming task by synthesizing a primer that contains a short 10 nucleotide stretch of RNA followed by 10 to 20 DNA bases.[3] Importantly, this priming action occurs at replication initiation at origins to begin leading-strand synthesis and also at the 5′ end of each Okazaki fragment on the lagging strand.

However, Pol α is not able to continue DNA replication and must be replaced with another polymerase to continue DNA synthesis.[67] Polymerase switching requires clamp loaders and it has been proven that normal DNA replication requires the coordinated actions of all three DNA polymerases: Pol α for priming synthesis, Pol ε for leading-strand replication, and the Pol δ, which is constantly loaded, for generating Okazaki fragments during lagging-strand synthesis.[68]

Cdc45–Mcm–GINS Helicase Complex[edit]

The DNA helicases and polymerases must remain in close contact at the replication fork. If unwinding occurs too far in advance of synthesis, large tracts of single-stranded DNA are exposed. This can activate DNA damage signaling or induce DNA repair processes. To thwart these problems, the eukaryotic replisome contains specialized proteins that are designed to regulate the helicase activity ahead of the replication fork. These proteins also provide docking sites for physical interaction between helicases and polymerases, thereby ensuring that duplex unwinding is coupled with DNA synthesis.[73]

Proliferating Cell Nuclear Antigen[edit]

Main article: proliferating cell nuclear antigen

To strengthen the interaction between the polymerase and the template DNA, DNA sliding clamps associate with the polymerase to promote the processivity of the replicative polymerase. In eukaryotes, the sliding clamp is a homotrimer ring structure known as the proliferating cell nuclear antigen (PCNA). The PCNA ring has polarity with surfaces that interact with DNA polymerases and tethers them securely to the DNA template. PCNA-dependent stabilization of DNA polymerases has a significant effect on DNA replication because PCNAs are able to enhance the polymerase processivity up to 1,000-fold.[85][86] PCNA is an essential cofactor and has the distinction of being one of the most common interaction platforms in the replisome to accommodate multiple processes at the replication fork, and so PCNA is also viewed as a regulatory cofactor for DNA polymerases.[87)

PCNA loading is accomplished by the replication factor C (RFC) complex. The RFC complex is composed of five ATPases: Rfc1, Rfc2, Rfc3, Rfc4 and Rfc5.[88] RFC recognizes primer-template junctions and loads PCNA at these sites.[89][90] The PCNA homotrimer is opened by RFC by ATP hydrolysis and is then loaded onto DNA in the proper orientation to facilitate its association with the polymerase.[91][92] Clamp loaders can also unload PNCA from DNA; a mechanism needed when replication must be terminated.[92]

Termination

The end replication problem is handled in eukaryotic cells by telomere regions and telomerase. Telomeres extend the 3′ end of the parental chromosome beyond the 5′ end of the daughter strand. This single-stranded DNA structure can act as an origin of replication that recruits telomerase. Telomerase is a specialized DNA polymerase that consists of multiple protein subunits and an RNA component. The RNA component of telomerase anneals to the single stranded 3′ end of the template DNA and contains 1.5 copies of the telomeric sequence.[60] Telomerase contains a protein subunit that is a reverse transcriptase called telomerase reverse transcriptase or TERT. TERT synthesizes DNA until the end of the template telomerase RNA and then disengages.[60] This process can be repeated as many times as needed with the extension of the 3′ end of the parental DNA molecule. This 3′ addition provides a template for extension of the 5′ end of the daughter strand by lagging strand DNA synthesis. Regulation of telomerase activity is handled by telomere-binding proteins.

-Working_principle_of_telomerase

 

A depiction of telomerase progressively elongating telomeric DNA.

 

DNA replication is a tightly orchestrated process that is controlled within the context of the cell cycle. Progress through the cell cycle and in turn DNA replication is tightly regulated by the formation and activation of pre-replicative complexs (pre-RCs) which is achieved through the activation and inactivation of cyclin-dependent kinases (Cdks). Specifically it is the interactions of cyclins and cyclin dependent kinases that are responsible for the transition from G1 into S-phase.

 

 

 

 

-Cell_Cycle_

Cell_Cycle_

 

 

 

 

 

 

 

Bhatt et al., GA, 6-26-12

 

Revised_definition_of_gene_and_flow_of_genetic_information

 

 

 

 

 

 

 

 

 

 

Epigenetic_mechanisms

 

– G-quadruplex

It will be exactly 60 years ago in February that James Watson and Francis Crick famously burst into the pub next to their Cambridge laboratory to announce the discovery of the “secret of life”.

What they had actually done was describe the way in which two long chemical chains wound up around each other to encode the information cells need to build and maintain our bodies.

Today, the pair’s modern counterparts in the university city continue to work on DNA’s complexities.

Balasubramanian’s group has been pursuing a four-stranded version of the molecule that scientists have produced in the test tube now for a number of years.

It is called the G-quadruplex. The “G” refers to guanine, one of the four chemical groups, or “bases”, that hold DNA together and which encode our genetic information (the others being adenine, cytosine, and thymine).

The G-quadruplex seems to form in DNA where guanine exists in substantial quantities.

And although ciliates, relatively simple microscopic organisms, have displayed evidence for the incidence of such DNA, the new research is said to be the first to firmly pinpoint the quadruple helix in human cells.

‘Funny target’

The team, led by Giulia Biffi, a researcher in Balasubramaninan’s lab, produced antibody proteins that were designed specifically to track down and bind to regions of human DNA that were rich in the quadruplex structure. The antibodies were tagged with a fluorescence marker so that the time and place of the structures’ emergence in the cell cycle could be noted and imaged.

This revealed the four-stranded DNA arose most frequently during the so-called “s-phase” when a cell copies its DNA just prior to dividing.

Prof Balasubramaninan said that was of key interest in the study of cancers, which were usually driven by genes, or oncogenes, that had mutated to increase DNA replication.

If the G-quadruplex could be implicated in the development of some cancers, it might be possible, he said, to make synthetic molecules that contained the structure and blocked the runaway cell proliferation at the root of tumours.

 

 

John Berger

Founder at Novagon DNA

If the first and core mission of the genetic code is to faithfully replicate the “genetic material” encoded in the DNA and RNA nucleic acids, then every metabolic process must be functioning in a synchronous 24/7 manner. The only way to do this is to use all the purine and pyrmidine nucleotide, nucleoside and bases (ATUIXGC) =7 necessary and sufficient to make RNA first and then with the assistance of Thioredoxin i.e. ferredoxin purple sulphur bacteria to oxidize rna to dna.

In regards to purine metabolism which is my major area of focus. The two purine nucleotides left out of the current genetic code i.e. IMP and XMP have the following functions through their enzymes.1. Begin purine nucleotide synthesis de novo by IMPDH cyclodehydrogenase the last step in closing the purine ring and the current foundation molecular structure for DNA and RNA; 2. HPRT is the main enzyme is purine salvage for IMP and GMP; APRT provides same service for AMP; 3. Finally the last step in purine metabolism is by xanthine oxidase with the assistance of FES and molybendum. In essence the IMP and XMP families were the first to build the nucleic acid molecular structure; design a process to recycle functional side groups while keeping the purine ring intact and finally developing the biochemical pathway to eliminate toxic ammonia NH3 from the CNS and liver/kidneys.

I believe the 7 nucleotide Novagon DNA triplex genetic code should be called the epigenetic code since it works not only in protein metabolism which is 2% of the genome but noncoding intronic regions ie. rna editing, RNAi, piRNA, snMRN, long noncoding RNA and many other small rnas which operate above the level of the dna and rna base pair i.e. epigenesis suppressing or enhancing whole genes and networks of genes which control protein,lipid,carbohydrate and nucleic acid metabolism.

I am in the process of deveoping a 7 code epigenetic primer to control the gene switches which in turn allows the genetic material to be inherited from generation to generation as the species constantly adapts to external and internal stressors and competitive antagonist.

A Conserved Structural Core in Type II Restriction Enzymes.

A Conserved Structural Core in Type II Restriction Enzymes.

 

 

 

 

Dna triplex pic

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Agents that Damage DNA

  • Certain wavelengths of radiation
    • ionizing radiation such as gamma rays and X-rays
    • ultraviolet rays, especially the UV-C rays (~260 nm) that are absorbed strongly by DNA but also the longer-wavelength UV-B that penetrates the ozone shield [Link].
  • Highly-reactive oxygen radicals produced during normal cellular respiration as well as by other biochemical pathways. [Link to further discussion.]
  • Chemicals in the environment
    • many hydrocarbons, including some found in cigarette smoke

  Aflatoxin structures

 

 

 

 

 

Link to description of a test measuring the mutations caused by the hydrocarbon benzopyrene.
    • some plant and microbial products, e.g. the aflatoxins produced in moldy peanuts
  • Chemicals used in chemotherapy, especially chemotherapy of cancers

Types of DNA Damage

  1. All four of the bases in DNA(A, T, C, G)can be covalently modified at various positions.
    • One of the most frequent is the loss of an amino group(“deamination”) — resulting, for example, in a C being converted to a U.
  2. Mismatchesof the normal bases because of a failure of proofreading during DNA replication.
    • Common example: incorporation of the pyrimidineU (normally found only in RNA) instead of T.
  3. Breaksin the backbone.
    • Can be limited to one of the two strands (a single-stranded break, SSB) or
    • on both strands(a double-stranded break (DSB).
    • Ionizing radiation is a frequent cause, but some chemicals produce breaks as well.
  4. CrosslinksCovalent linkagescan be formed between bases
    • on the same DNA strand (“intrastrand”) or
    • on the opposite strand (“interstrand”).

Several chemotherapeutic drugs used against cancers crosslink DNA [Link].

Repairing Damaged Bases

Damaged or inappropriate bases can be repaired by several mechanisms:

  • Direct chemical reversal of the damage
  • Excision Repair, in which the damaged base or bases are removed and then replaced with the correct ones in a localized burst of DNA synthesis. There are three modes of excision repair, each of which employs specialized sets of enzymes.
    1. Base Excision Repair (BER)
    2. Nucleotide Excision Repair (NER)
    3. Mismatch Repair (MMR)

 

 

Gene expression profiles associated with acute myocardial infarction and risk of cardiovascular death

J Kim,  N Ghasemzadeh,  DJ Eapen, NC Chung, JD Storey, AA Quyyumi and G Gibson
Kim et al. Genome Medicine 2014, 6:40
http://genomemedicine.com/content/6/5/40

Background: Genetic risk scores have been developed for coronary artery disease and atherosclerosis, but are not predictive of adverse cardiovascular events. We asked whether peripheral blood expression profiles may be predictive of acute myocardial infarction (AMI) and/or cardiovascular death.

Methods: Peripheral blood samples from 338 subjects aged 62 ± 11 years with coronary artery disease (CAD) were analyzed in two phases (discovery N = 175, and replication N = 163), and followed for a mean 2.4 years for cardiovascular death. Gene expression was measured on Illumina HT-12 microarrays with two different normalization procedures to control technical and biological covariates. Whole genome genotyping was used to support comparative genome-wide association studies of gene expression. Analysis of variance was combined with receiver operating curve and survival analysis to define a transcriptional signature of cardiovascular death.

Results: In both phases, there was significant differential expression between healthy and AMI groups with overall down-regulation of genes involved in T-lymphocyte signaling and up-regulation of inflammatory genes. Expression quantitative trait loci analysis provided evidence for altered local genetic regulation of transcript abundance in AMI samples. On follow-up there were 31 cardiovascular deaths. A principal component (PC1) score capturing covariance of 238 genes that were differentially expressed between deceased and survivors in the discovery phase significantly predicted risk of cardiovascular death in the replication and combined samples (hazard ratio = 8.5, P< 0.0001) and improved the C-statistic (area under the curve 0.82 to 0.91, P= 0.03) after adjustment for traditional covariates.

Conclusions: A specific blood gene expression profile is associated with a significant risk of death in Caucasian subjects with CAD. This comprises a subset of transcripts that are also altered in expression during acute myocardial infarction.

 

Lecture Contents delivered at Koch Institute for Integrative Cancer Research, Summer Symposium 2014: RNA Biology, Cancer and Therapeutic Implications, June 13, 2014 @MIT

Curator of Lecture Contents: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.wordpress.com/wp-admin/post.php?post=23174&action=edit
3:15 – 3:45, 6/13/2014, Laurie Boyer “Long non-coding RNAs: molecular regulators of cell fate”     https://pharmaceuticalintelligence.com/2014/06/13/315-345-2014-laurie-boyer-long-non-coding-rnas-molecular-regulators-of-cell-fate/

 

TAR DNA-binding protein 43

TDP-43 is a transcriptional repressor that binds to chromosomally integrated TAR DNA and represses HIV-1 transcription. In addition, this protein regulates alternate splicing of the CFTR gene. In particular, TDP-43 is a splicing factor binding to the intron8/exon9 junction of the CFTR gene and to the intron2/exon3 region of the apoA-II gene.[2] A similar pseudogene is present on chromosome 20.[3]

TDP-43 has been shown to bind both DNA and RNA and have multiple functions in transcriptional repression, pre-mRNA splicing and translational regulation.

TDP-43 was originally identified as a transcriptional repressor that binds to chromosomally integrated trans-activation response element (TAR) DNA and represses HIV-1 transcription.[1] It was also reported to regulate alternate splicing of theCFTR gene and the apoA-II gene.

In spinal motor neurons TDP-43 has also been shown in humans to be a low molecular weight microfilament (hNFL) mRNA-binding protein.[4] It has also shown to be a neuronal activity response factor in the dendrites of hippocampal neurons suggesting possible roles in regulating mRNA stability, transport and local translation in neurons.[5]

Clinical significance[edit]

Hyper-phosphorylatedubiquitinated and cleaved form of TDP-43, known as pathologic TDP43, is the major disease protein in ubiquitin-positive, tau-, and alpha-synuclein-negative frontotemporal dementia (FTLD-TDP, previously referred to as FTLD-U[6]) and in Amyotrophic lateral sclerosis (ALS).[7] Elevated levels of the TDP-43 protein have also been identified in individuals diagnosed with chronic traumatic encephalopathy, a condition that often mimics ALS and that has been associated with athletes who have experienced multiple concussions and other types of head injury.[8]

HIV-1, the causative agent of acquired immunodeficiency syndrome (AIDS), contains an RNA genome that produces a chromosomally integrated DNA during the replicative cycle. Activation of HIV-1 gene expression by the transactivator “Tat” is dependent on an RNA regulatory element (TAR) located “downstream” (i.e. to-be transcribed at a later point in time) of the transcription initiation site.

Mutations in the TARDBP gene are associated with neurodegenerative disorders including frontotemporal lobar degeneration and amyotrophic lateral sclerosis (ALS).[9] In particular, the TDP-43 mutants M337V and Q331K are being studied for their roles in ALS.[10][11] Cytoplasmic TDP-43 pathology is the dominant histopathological feature of multisystem proteinopathy.[12]

 

 

General annotation (Comments)

Function DNA and RNA-binding protein which regulates transcription and
splicing. Involved in the regulation of CFTR splicing. It promotes
CFTR exon 9 skipping by binding to the UG repeated motifs in the
polymorphic region near the 3′-splice site of this exon. The resulting
aberrant splicing is associated with pathological features typical of
cystic fibrosis. May also be involved in microRNA biogenesis,
apoptosis and cell division. Can repress HIV-1 transcription by
binding to the HIV-1 long terminal repeat. Stabilizes the low
molecular weight neurofilament (NFL) mRNA through a direct
interaction with the 3′ UTR. Ref.2 Ref.12
Subunit structure Homodimer. Interacts with BRDT By similarity. Binds specifically to
pyrimidine-rich motifs of TAR DNA and to single stranded TG
repeated sequences. Binds to RNA, specifically to UG repeated
sequences with a minimun of six contiguous repeats. Interacts with
ATNX2; the interaction is RNA-dependent. Ref.16
Subcellular location Nucleus. Note: In patients with frontotemporal lobar degeneration
and amyotrophic lateral sclerosis, it is absent from the nucleus of
affected neurons but it is the primary component of cytoplasmic
ubiquitin-positive inclusion bodies. Ref.2 Ref.11
Tissue specificity Ubiquitously expressed. In particular, expression is high in pancreas,
placenta, lung, genital tract and spleen.
Domain The RRM domains can bind to both DNA and RNA By similarity.
Post-translational modification Hyperphosphorylated in hippocampus, neocortex, and spinal cord
from individuals affected with ALS and FTLDU. Ref.11Ubiquitinated in hippocampus, neocortex, and spinal cord from
individuals affected with ALS and FTLDU. Ref.2 Ref.11  Cleaved to
generate C-terminal fragments in hippocampus, neocortex, and
spinal cord from individuals affected with ALS and FTLDU.
Involvement in disease Amyotrophic lateral sclerosis 10 (ALS10) [MIM:612069]: A
neurodegenerative disorder affecting upper motor neurons in the
brain and lower motor neurons in the brain stem and spinal cord,
resulting in fatal paralysis. Sensory abnormalities are absent. The
pathologic hallmarks of the disease include pallor of the corticospinal
tract due to loss of motor neurons, presence of ubiquitin-positive
inclusions within surviving motor neurons, and deposition of
pathologic aggregates. The etiology of amyotrophic lateral sclerosis is likely to be multifactorial, involving both genetic and environmental factors. The disease is inherited in 5-10% of the cases.  Note: The disease is caused by mutations affecting the gene represented in this
entry.

  1. 16Ref.21 Ref.22 Ref.23 Ref.24 Ref.25 Ref.26 Ref.27 Ref.28 Ref.29 Ref.30 Ref.31Ref.32
Sequence similarities Contains 2 RRM (RNA recognition motif) domains.

 

 

How DNA is made?

Deoxyribonucleic acid (DNA) synthesis is a process by which copies of nucleic acid strands are made. In nature, DNA synthesis takes place in cells by a mechanism known as DNA replication. Using genetic engineering and enzyme chemistry, scientists have developed man-made methods for synthesizing DNA. The most important of these is poly-merase chain reaction (PCR). First developed in the early 1980s, PCR has become a multi-billion dollar industry with the original patent being sold for $300 million dollars.

History

DNA was discovered in 1951 by Francis Crick, James Watson, and Maurice Wilkins. Using x-ray crystallography data generated by Rosalind Franklin, Watson and Crick determined that the structure of DNA was that of a double helix. For this work, Watson, Crick, and Wilkins received the Nobel Prize in Physiology or Medicine in 1962. Over the years, scientists worked with DNA trying to figure out the “code of life.” They found that DNA served as the instruction code for protein sequences. They also found that every organism has a unique DNA sequence and it could be used for screening, diagnostic, and identification purposes. One thing that proved limiting in these studies was the amount of DNA available from a single source.

After the nature of DNA was determined, scientists were able to examine the composition of the cellular genes. A gene is a specific sequence of DNA base pairs that provide the code for the construction of a protein. These proteins determine the traits of an organism, such as eye color or blood type. When a certain gene was isolated, it became desirable to synthesize copies of that molecule. One of the first ways in which a large amount of a specific DNA was synthesized was though genetic engineering.

Genetic engineering begins by combining a gene of interest with a bacterial plasmid. A plasmid is a small stretch of DNA that is found in many bacteria. The resulting hybrid DNA is called recombinant DNA. This new recombinant DNA plasmid is then injected into bacterial cells. The cells are then cloned by allowing it to grow and multiply in a culture. As the cells multiply so do copies of the inserted gene. When the bacteria has multiplied enough, the multiple copies of the inserted gene can then be isolated. This method of DNA synthesis can produce billions of copies of a gene in a couple of weeks.

In 1983, the time required to produce copies of DNA was significantly reduced when Kary Mullis developed a process for synthesizing DNA called polymerase chain reaction (PCR). This method is much faster than previous known methods producing billions of copies of a DNA strand in just a few hours. It begins by putting a small section of double stranded DNA in a solution containing DNA polymerase, nucleotides and primers. The solution is heated to separate the DNA strands. When it is cooled, the polymerase creates a copy of each strand. The process is repeated every five minutes until the desired amount of DNA is produced. In 1993, Mullis’s development of PCR earned him the Nobel Prize in Chemistry.

Background

The key to understanding DNA synthesis is understanding its structure. Typically, DNA exists as two chains of chemically linked nucleotides. These links follow specific patterns dictated by the base pairing rules. Each nucleotide is made up of a deoxyribose sugar molecule, a phosphate group, and one of four nitrogen containing bases. The bases include the pyrimidines thymine (T) and cytosine (C)and the purines adenine (A) and guanine (G). In DNA, adenine generally links with thymine and guanine with cytosine. The molecule is arranged in a structure called a double helix which can be imagined by picturing a twisted ladder or spiral staircase. The bases make up the rungs of the ladder while the sugar and phosphate portions make up the ladder sides. The order in which the nucleotides are linked, called the sequence, is determined by a process known as DNA sequencing.

In a eukaryotic cell, DNA synthesis occurs just prior to cell division through a process called replication. When replication begins the two strands of DNA are separated by a variety of enzymes. Thus opened, each strand serves as a template for producing new strands. This whole process is catalyzed by an enzyme called DNA polymerase. This molecule brings corresponding, or complementary, nucleotides in line with each of the DNA strands. The nucleotides are then chemically linked to form new DNA strands which are exact copies of the original strand. These copies, called the daughter strands, contain half of the parent DNA molecule and half of a whole new molecule. Replication by this method is known as semiconservative replication.

Raw Materials

The primary raw materials used for DNA synthesis include DNA starting materials, taq DNA polymerase, primers, nucleotides, and the buffer solution. Each of these play an important role in the production of millions of DNA molecules.

Controlled DNA synthesis begins by identifying a small segment of DNA to copy. This is typically a specific sequence of DNA that contains the code for a desired protein. Called template DNA, this material must be highly purified.

While the process of DNA replication was known before 1980, PCR was not possible because there were no known heat stable DNA polymerases.  In the early 1980s, scientists found bacteria living around natural steam vents. It turned out that these organisms, called thermus aquaticus, had a DNA polymerase that was stable and functional at extreme levels of heat. This taq DNA polymerase became the cornerstone for modern DNA synthesis techniques. During a typical PCR process, 2-3 micrograms of taq DNA polymerase is needed.

The polymerase builds the DNA strands by combining corresponding nucleotides on each DNA strand. Chemically speaking, nucleotides are made up of three types of molecular groups including a sugar structure, a phosphate group, and a cyclic base. The sugar portion provides the primary structure for all nucleotides. In general, the sugars are composed of five carbon atoms with a number of hydroxy (-OH) groups attached. For DNA, the sugar is 2-deoxy-D-ribose. The defining part of a nucleotide is the hetero-cyclic base that is covalently bound to the sugar. These bases are either pyrimidine or purine groups, and they form the basis for the nucleic acid code. Two types of purine bases are found including adenine and guanine. In DNA, two types of pyrimidine bases are present, thymine and cytosine. A phosphate group makes up the final portion of a nucleotide. This group is derived from phosphoric acid and is covalently bonded to the sugar structure on the fifth carbon.

cost of oligo and gene synthesis

 

 

 

 

The first phase of polymerase chain reaction (PCR) involves the denaturation of DNA. This “opening up” of the DNA molecule provides the template for the next DNA molecule from which to be produced. With the DNA split into separate strands, the temperature is lowered—the primer annealing step. During the next phase, the DNA polymerase interacts with the strands and adds complementary nucleotides along the entire length. The time required at this phase is about one minute for every 1,000 base pairs.

To initiate DNA synthesis, short primer sections of DNA must be used. These primer sections, called oligo fragments, are about 18-25 nucleotides in length and correspond to a section on the template DNA. They typically have a C and G nucleotide concentration of about 60% with even distribution. This provides the maximum efficiency in the synthesis process.

The buffer solution provides the medium in which DNA synthesis can occur. This is an aqueous solution which contains MgCl2, HCI, EDTA, and KCI. The MgCl2 concentration is important because the Mg2+ ions interact with the DNA and the primers creating crucial complexes for DNA synthesis. The pH of this system is critical so it may also be buffered with ammonium sulfate. To energize the reaction, various energy molecules are added such as ATP, GTP, and NTP.

DNA synthesis involves three distinct processes, typically done in separate areas to avoid contamination, including sample preparation, DNA synthesis reaction cycle and DNA isolation. Following these procedures scientists are able to convert a few strands of DNA into millions and millions of exact copies.

Preparation of the samples

  • 1 Typically, all of the starting solutions except the primers, polymerases and the dNTPs are put in an autoclave to kill off any contaminating organism. Two separate solutions are made. One contains the buffer, primers and the polymerase. The other contains the MgCl2 and the template DNA. These solutions are all put into small tubes to begin the reaction.

 

Kary Banks Mullis.

Kary Banks Mullis was born in Lenoir, North Carolina, in 1944. Upon graduation from Georgia Tech in 1966 with a B.S. in chemistry, Muilis entered the biochemistry doctoral program at the University of California, Berkeley. Earning his Ph.D. in 1973, he accepted a teaching position at the University of Kansas Medical School in Kansas City. In 1977, he assumed a postdoctoral fellowship at the University of California, San Francisco.

Muilis accepted a position as a research scientist in 1979 with a growing biotech firm—Cetus Corporation, in Emeryville, California—that synthesized chemicals used by other scientists in genetic cloning. While there, he designed polymerase chain reaction (PCR), a fast and effective technique for reproducing specific genes or DNA (deoxyribonucleic acid) fragments that can create billions of copies in a few hours. The most effective way to reproduce DNA was by cloning, but it was problematic. It took time to convince Mullis’s colleagues of the importance of this discovery but soon PCR became the focus of intensive research. Scientists at Cetus developed a commercial version of the process and a machine called the Thermal Cycler (with the addition of the chemical building blocks of DNA [nucleotides] and a biochemical catalyst [polymerase], the machine would perform the process automatically on a target piece of DNA).

Read more: http://www.madehow.com/Volume-6/DNA-Synthesis.html#ixzz38sovuX5n

types_RNAi_Q3_used_in_research

 

lncRNA-s   A summary of the various functions described for lncRNA

 

Additional References to Leaders in Pharmaceutical Intelligence

Content Consultant: Larry H Bernstein, MD, FCAP

Series C: e-Books on Cancer & Oncology

Content Consultant: Larry H Bernstein, MD, FCAP.

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Pharmacogenomics needs new materials that are inert against the host and specifically  active to modulate molecular metabolism towards wanted homeostasis of the physiological system.  These can come from natural resources or men-made.  That is why we must know the origin  to  improve.     Recently, Synthetic Biology, even though it is a developing upcoming field, it is generating mile stones for applications in the clinic, the biotechnology industry and in basic molecular research. As  a result, it created a multidisciplinary expertise from scientists to engineers.  Among other things extending the search to first life on Earth is one of the many alternatives.  Here I like to present how synthetic biology can be initiated onto Translational Medicine from adiscovery of molecules from the sea.

Microorganisms played a role in evolution to start a life.  99 % of our genome is related to microbial organisms. initially there was a classical  Microbiology, then evolved to Industrial Microbiology and Biotechnology then Microbial Genomics and now Microbiome and Health became the focus.  Finally,  the circle is getting tide into how microbiome involved with healthy and disease state of human? How they can be used that is what it really means to include microorganisms into human health for diagnostics and targeted therapies?

Or should we start from  scarcity?

Microbiology is my first formal education and  building block.  Simple but help to understand system biology and  the mechanism of life in a nut shell.   The closest field is embryonic stem cell biology for building “synthesizing” a whole new organism.  Then  system biology and developmental biology also gain interest.

The real  remember the month of October in 2001 when DOE reported that they sequenced 23 organisms in Walnut Creek.  Having seen presentation to identify microorganisms through complex crystal structure assays through chemical pathway  at the Microbial Genomics Meeting organized by ASM in Monterey, CA in 2001.

Discovery of microorganisms in marine life like in Mediterranean Sea, containing 38% salt,is very similar with finding circulating disease making cells.   Yet, they are similar since both search for a specific needle in the pile.  Furthermore, the unique behavior of enzymes from microbial organisms such as Taq polymerase or restriction enzymes made it possible for us to develop new technologies for copying and propagating significant sequences.  When these early molecular biology methods are combined with the power of genomics and knowledge of unique structures in molecular physiology, it is possible to design better and sensitive sensors or build an organism to rptect or fix the need of the body.  neither sensors nor synthesized organism model are complete since one is missing the basic element of life “transformation of information” the other is missing the integrity that once nature provided in a single simple cell.

Having sensory smart chip/band/nanomolecule to redesign the cells may also possible if only we know the combination.  Thus, we have options to deliver if we know what to be carried.

An external file that holds a picture, illustration, etc.<br /><br /><br /><br /><br />
Object name is marinedrugs-11-00700-g002.jpg

(Figure: The combined strategy of gene-based screening and bioactivity-based screening for marine microbial natural products (MMNPs) discovery, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3705366/figure/marinedrugs-11-00700-f002/)

As we come across, novel pathways or primary pathways of physiology gain significant interest to determine marine microbial compound for therapeutics since they are further away from the evolution three that gives an advantage for biomedical/translational scientist to avoid most part of th eimmune responses such as inflammation, toxicity. Yes, indeed these are not scientific tails but true since currently, 16 of 20 marine antitumor compounds under clinical trial are derived from microbial sources because marine microorganisms are a major source for MMNP discovery.  However, isolation of these organisms.  For example, pretreatment methods, enrichment, physical, and chemical techniques (e.g., dry heat, exposure to 1%–1.5% phenol, sucrose-gradient centrifugation, and filtration through cellulose membrane filters) can be applied to increase especially the less abundant specific groups of marine microorganisms, . A variety of pretreatment methods including recovery of these microorganisms.  This reminds me ecosystem of the soil, since in soil the trouble is identifying the specific culture among millions of others.

Regardless of the case,  nutrients are the key for selecting and isolating any organisms but specifically, as a result any marine microbes have specific nutrient requirements for growth (e.g., sponge extract ) or chemical (e.g., siderophores, signal molecules, non-traditional electron donors, and electron acceptors.  This also should remind us subject of Biology 101 Essential Vitamins and Minerals.  What we eat who we are.

For example, Bruns et al. employed technique where they employed different carbon substrates (agarose, starch, laminarin, xylan, chitin, and glucose) at low concentrations (200 μM each) so that they can  improve the cultivation efficiency of bacteria from the Gotland Deep in the central Baltic Sea. As a result of this growth medium they were able to elevate yield, which is created higher cultivation efficiencies (up to 11% in fluid media) compared to other studies.

Yet, another component must be addressed that is culture medium such as ionic strength for a microbila growth. For example, Tsueng et al. study on marine actinomycete genus Salinispora that can produce bioactive secondary metabolites such as desferrioxamine, saliniketals, arenamides, arenimycin and salinosporamide.  However, they observed that  three species of SalinisporaS. arenicolaS. tropica, and S. pacifica require a high ionic strength but  S. arenicolahas a lower growth requirement for ionic strength than S. tropica and S. pacificaUsing after assaying them against sodium chloride-based and lithium chloride-based media. As  aresult, there is a specificity for growth. 

In addition, energy must be supported imagine that in marine organisms the metabolism is very unique, may be slow and possibly.  However, the main criteria is  most of them grow under low oxygen conditions like tumors.  Warburg effect posed a  problem for human but helped microorganisms to survive and evolve.  One’s weakness the other’s strength make a great teamwork for solving diseases of human kind es especially for cancer. 

This reminds us to utilize minerals, electrons specifically after all the simplest form of carbon metabolism based on biochemical pathways like Crebs cycle, one carbon metabolism and amino acid metabolism etc. Even though 90% of human body made up off microbial origin there are microorganisms that are not cultured yet.

The irony is less than 1% of microorganisms can be cultured.  Furthermore, they are not included for representing the total phylogenetic diversity. Therefore, majority of work concentrated on finding and cultivating the uncultured majority of the microbial world for MMNPs’.  For example,  an uncultivated bacterial symbiont of the marine sponge Theonella swinhoei  producing many antitumor compounds such as pederin, mycalamide A, and onnamide A.

In any conditions if any living needs to be recognized and remembered, their place would be either on top or the bottom of the stack. Microbiome searches for specificity among tone of other organisms to recognize the disease, changes in cell differentiation and pathways or marine microbiologist search for uncommon scarce organisms. Yet, both of them are beneficial with their unique way.

Then what is the catch or fuss?  The catch is screening to identify what makes this organism unique that can be use for human health. Translational medicine may start from the beginning of life from microorganisms created.  This can be called with its newly coined named”synthetic biology” but if we go further than the conventional screening methods which include bioactivity-guided screening and gene-guided screening  and increase the power with genomics we may call it “synthetic genomics”.

As  a result these signature sequences establishes the “unique” biomarkers  or therpaeutics to be used for drug discovery, making vaccines, and remodulating the targeted cells. How?

These microorganisms secrete these metabolites or proteins to their growth medium just like a soluble protein, if you will like a inflammation factor or any other secreted protein of our human body cells. Collecting substrate or extract the pellet could be the choice.   in a nut shell this require at least three steps: First, finding the bioactivity, apply bioactivity-guided screening for direct detection of  the activity such as antimicrobial, antitumor, antiviral, and antiparasitic activities.  Second, a bioinformatic assessment of the secondary metabolite biosynthetic potential in the absence of fully assembled pathways or genome sequences. Third, application on cell lines and possible onto model organisms can improve the process of MMNP discovery so that allow us to prioritize strains for fermentation studies and chemical analysis. 

In summary, establish the culture growth, analyze bioactivity and discover the new gene product to be used.  Here is an example, first they  isolated Marinispora sp from the saline culture.  Next step,  identify new sources of bioactive secondary metabolites, gene-guided screening has been deployed to search target genes associated with NPs biosynthetic pathways, e.g., the fragments between ketosynthase and methylmalonyl-CoA transferase of polyketides (PKS) type I, enediyne PKS ketosynthase gene, O-methyltransferase gene, P450 monooxygenase gene, polyether epoxidase gene, 3-hydroxyl-3-methylglutaryl coenzyme A reductase gene, dTDP-glucose-4,6-dehydratase (dTGD) gene, and halogenase gene. The, apply bioinformatics that includes synthesizing the knowledge with  homology-based searches and phylogenetic analyses, gene-based screening  to predict new secondary metabolites discovered by isolates or environments.  Finally, identify the sequnce for PCR and use against a cell line or model organisms. In this case,  CNQ-140 based on significant antibacterial activities  against drug-resistant pathogens (e.g., MRSA) and impressive and selective cancer cell cytotoxicities (0.2–2.7 μM of MIC50 values) against six melanoma cell lines provided significant outcome. They are recognized as antitumor antibiotics with a new structural class, marinomycins A–D

This is a great method but there are two botle necks: 1. 99% of microbial organisms are not cultured in the labs. 2. Finding the optimum microbial growth and screening takes time. Thus, assesments can me done through metagenomics.  However, metagenomics has its shortcomings since on face of living unless applications applied in vivo in vitro results may not be valid.  The disadvantage of  metagenomics can be listed as:  1. inability of efficient acquisition of intact gene fragment,  2. incompatibility of expression elements such as promoter in a heterologous host.  On the pther hand, there can be possible resolution to avoid these factors  so metagenomics-based MMNP discovery can be plausable such as development  in  synthetic biology by large DNA fragment assembly techniques for artificial genome synthesis and synthetic microbial chassis suitable for different classes of MMNP biosynthesis.

However, many gene clusters have been identified by combined power of genomics and biioinformatics for MNP discovery.  This is  mainly necessary since  secondary metabolites usually biosynthesized by large multifunctional synthases that acts in a sequential assembly lines like adding carboxylic acid and amino acid building blocks into their products.  

 References

Simmons TL, Coates RC, Clark BR, Engene N, Gonzalez D, Esquenazi E, Dorrestein PC, Gerwick W

Proc Natl Acad Sci U S A. 2008 Mar 25; 105(12):4587-94.

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Reporter: Aviva Lev-Ari, PhD, RN

J Cardiovasc Transl Res. 2012 Sep 7. [Epub ahead of print]

Next Generation Diagnostics in Inherited Arrhythmia Syndromes : A Comparison of Two Approaches.

Ware JSJohn SRoberts AMBuchan RGong SPeters NSRobinson DOLucassen ABehr ERCook SA.

Source

MRC Clinical Sciences Centre, Imperial College London, London, UK, j.ware@imperial.ac.uk.

Abstract

Next-generation sequencing (NGS) provides an unprecedented opportunity to assess genetic variation underlying human disease. Here, we compared two NGS approaches for diagnostic sequencing in inherited arrhythmia syndromes. We compared PCR-based target enrichment and long-read sequencing (PCR-LR) with in-solution hybridization-based enrichment and short-read sequencing (Hyb-SR). The PCR-LR assay comprehensively assessed five long-QT genes routinely sequenced in diagnostic laboratories and “hot spots” in RYR2. The Hyb-SR assay targeted 49 genes, including those in the PCR-LR assay. The sensitivity for detection of control variants did not differ between approaches. In both assays, the major limitation was upstream target capture, particular in regions of extreme GC content. These initial experiences with NGS cardiovascular diagnostics achieved up to 89 % sensitivity at a fraction of current costs. In the next iteration of these assays we anticipate sensitivity above 97 % for all LQT genes. NGS assays will soon replace conventional sequencing for LQT diagnostics and molecular pathology.

PMID: 22956155 [PubMed]
Source: 
http://www.ncbi.nlm.nih.gov/pubmed/22956155

Researchers in the UK have compared a PCR-based and a capture hybridization-based assay for sequencing panels of inherited cardiovascular disease genes and have found both to be suitable for diagnostics in principle, though their sensitivity needs to be optimized.

According to James Ware, a clinical lecturer at Imperial College London, the purpose of the study, published online this month in the Journal of Cardiovascular Translational Research, was to evaluate different approaches for sequencing cardiovascular disease genes, both for molecular diagnosis and for large-scale resequencing research studies.

His group, in the National Institute for Health Research Royal Brompton Cardiovascular Biomedical Research Unit, is interested in a range of inherited heart disease types, including cardiomyopathies and inherited arrhythmia syndromes such as long QT syndrome.

For their study, they compared two next-gen sequencing assays: a PCR-based approach that uses Fluidigm’s Access Array to amplify 96 amplicons in five LQT genes and one other gene, followed by sequencing on the 454 GS Junior; and an in-solution hybridization approach that uses Agilent’s SureSelect to target 49 inherited arrhythmia genes and sequences them on Life Technologies’ SOLiD 4.

The study focused on the sensitivity of the assays, or how well they were able to capture their intended targets, rather than their specificity, or their ability to avoid false positives.

Ware said that at the time of the study, PCR and in-solution capture were the two main target selection methods available. The researchers are still using both approaches but are now employing “a wide range of sequencers” from various providers for both types of assays, including Illumina instruments and Life Tech’s Ion Torrent.

For their comparison, they analyzed 48 samples, of which they sequenced 33 with both approaches and 15 using either one or the other.

The samples included 19 known variants in three disease genes, of which the hybridization-SOLiD method detected 17 and the PCR-454 method 14. Undetected variants were generally in areas that were not well covered, either due to a failure in enrichment, sequencing, or because the alignment was not unique. One variant that was missed by both approaches fell in a very GC-rich region.

Consumables costs for both assays were considerably lower than with Sanger sequencing: While sequencing five genes by Sanger costs more than $700 in consumables, the five-gene PCR/454 assay cost about $55 and the 49-gene hybridization/SOLiD assay cost about $200, according to the study.

Turnaround time is the shortest for Sanger sequencing, which, according to the study, can be done in one day for five genes and 17 samples, not including sample prep. The PCR/454 assay takes about two days for target enrichment and sequencing 48 samples, and the hybridization/SOLiD assay takes about two weeks for sequencing alone, they wrote.

Overall, Ware said, both sequencing approaches performed “reasonably well” and are significantly cheaper than Sanger sequencing. He said that in the UK, molecular diagnosis for inherited cardiovascular disease has traditionally been performed by Sanger, at a cost of approximately £500 to £1,000 ($800 to $1,600) for several genes involved in a clinical condition. However, for cost reasons, not all relevant genes are usually sequenced.

Target selection was the performance-limiting step for both approaches, a result the researchers expected. “It sounds obvious, but not all genes are equally easy to target,” Ware said. For example, in the hybridization assay, the overall target coverage was about 98 percent, but for some genes, it was only 80 percent or 90 percent. The two most important genes in long QT syndrome, KCNQ1 and KCNH2, “proved to be the hardest to sequence.”

Thus, for diagnostic use of NGS gene panels, “it’s important to know not just how the system performs overall but really how it’s performing for the specific genes you’re interested in,” he said.

To use either approach in diagnostics, the target selection step would need to be optimized. Ware’s team has already improved both assays and is now trying them in a number of fully Sanger-sequenced samples to study both sensitivity and specificity.

Longer term, the sensitivity of next-gen sequencing could approach that of Sanger sequencing, he said. And even if it does not reach 100 percent, because NGS approaches can target so many more genes, “maybe you can afford a very slight tradeoff in the per-gene sensitivity if the overall diagnostic sensitivity of the panel goes up,” he said. “At the moment, because we don’t have that much experience in sequencing the less-common genes, we don’t exactly know where that tradeoff lies.” In addition, any gaps could be filled by Sanger sequencing, while the test would probably still be cost effective.

Each approach also has some features that make it more suitable for certain applications. The PCR-based method has a fast turnaround and an “extremely user-friendly workflow,” Ware said, but it can only accommodate a small number of genes at the moment. His team also found it to be easier to optimize and improve. Thus, in the short term, PCR and sequencing “is probably closer to providing a diagnostic solution,” he said, especially for conditions where only a few genes are causative.

The hybridization-based approach, on the other hand, has much greater capacity, and there are advantages in “having a single assay that covers everything,” he said. It might also be possible to detect copy number variants using this approach, but not the more limited PCR method, he added.

Ware and his colleagues are currently using the hybridization approach to study a large panel of genes in 2,000 well-phenotyped volunteers, both healthy individuals and heart disease patients.

They have also started to use the hybridization method to sequence the TTN gene, truncating mutations in which were recently found to be a common cause of dilated cardiomyopathy. They are running the TTN test routinely for patients consented for research diagnostic testing that is not available anywhere else. Because this gene is so large, it is “completely impractical to be sequenced by conventional Sanger,” Ware said.

Julia Karow tracks trends in next-generation sequencing for research and clinical applications for GenomeWeb’s In Sequenceand Clinical Sequencing News. E-mail her here or follow her GenomeWeb Twitter accounts at @InSequence and@ClinSeqNews.

 

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Molecular pathogen identification comes to the bedside

Reporter:  Larry H Bernstein, MD, FCAP

The developments in molecular diagnostics have been proceeding at a rapid pace.  Naturally it is not surprising that it would reach into clinical microbiology early.  Microbiology and virology have many methods for validation of type of pathogen, and the identification of new pathogens can require delay because of use of a State laboratory.  This will be less an issue with the consolidation of regional facilities and associated laboratories.

I present an example of point-of-care technology from the University of California, Davis developed by Gerald Kost and colleagues with UC Lawrence Livermore National Point-of-Care Technologies Center .

Tran NK, Wisner DH, Albertson TE, Cohen S, et al.  Multiplex polymerase chain reaction pathogen detection in patients with suspected septicemia after trauma, emergency, and burn surgery. Surgery 2012 Mar;151(3):456-63. Epub 2011 Oct 5.  nktran@ucdavis.edu

The goal of the study:  to determine the clinical value of multiplex polymerase chain reaction (PCR) study for enhancing pathogen detection in patients with suspected septicemia after trauma, emergency, and burn surgery.

Finding: PCR-based pathogen detection quickly reveals occult bloodstream infections in these high-risk patients and may accelerate the initiation of targeted antimicrobial therapy.

Type study: a prospective observational study

Population:  30 trauma and emergency surgery patients compared to 20 burn patients.

Method:  Whole- routine blood cultures (BCs) were tested using a new multiplex, PCR-based, pathogen detection system. PCR results were compared to culture data.

Arbitrated Case Review

Arbitrated case review was performed by a medical intensivist, 3 trauma surgeons, 3 burn surgeons, 1 microbiologist, and an infectious disease physician to determine antimicrobial adequacy based on paired PCR/BC results. The arbitrated case review process is adapted from a previous study. Physicians were first presented cases with only BC results. Cases were then represented with PCR results included.

Results:

  • PCR detected rapidly more pathogens than culture methods.
  • Acute Physiology and Chronic Health Evaluation II (APACHE II), Sequential Organ Failure Assessment (SOFA), and Multiple Organ Dysfunction (MODS) scores were greater in PCR-positive versus PCR-negative trauma and emergency surgery patients (P ≤ .033).
  • Negative PCR results (odds ratio, 0.194; 95% confidence interval, 0.045-0.840; P = .028) acted as an independent predictor of survival for the combined surgical patient population.

CONCLUSION:

  • PCR results were reported faster than blood culture results.
  • Severity scores were significantly greater in PCR-positive trauma and emergency surgery patients.
  • The lack of pathogen DNA as determined by PCR served as a significant predictor of survival in the combined patient population.
  • PCR testing independent of traditional prompts for culturing may have clinical value in burn patients.

NK Tran, et al.  Multiplex Polymerase Chain Reaction Pathogen Detection in Trauma, Emergency, and Burn Surgery Patients with Suspected Septicemia.  Surgery. 2012 March; 151(3): 456–463. PMID: 21975287 [PubMed – indexed for MEDLINE] PMCID: PMC3304499 On-line 2011 October 5.
doi:  10.1016/j.surg.2011.07.030
PMCID: PMC3304499.  NIHMSID: NIHMS288960

Plymerase chain reaction, PCR

Plymerase chain reaction, PCR (Photo credit: Wikipedia)

 

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Author and Reporter: Ritu Saxena, Ph.D.

On 5th of July, I  discussed a general overview of varied mitochondrial functions, diseases, diagnosis and the current research focused on treatment of mitochondrial diseases in a post titled “Mitochondria: More than just the powerhouse of the cell”. https://pharmaceuticalintelligence.com/2012/07/09/mitochondria-more-than-just-the-powerhouse-of-the-cell/

Current post talks about a new technique that has been introduced by the authors as a ‘Comprehensive 1-Step Molecular Analyses of Mitochondrial Genome by Massively Parallel Sequencing’. The technique was recently published in the Clinical Chemistry journal (2012) by Zhang et al.

One mitochondria may have multiple copies of mtDNA  and an interesting feature observed in mitochondria is the heteroplasmy, a phenomenon where mutant and wild-type mtDNA can co-exist. During cell division, the mutant and wild-type copies are distributed randomly in daughter cells. The impact is in the heterogeneity with respect to penetrance and expressivity along that has diverse manifestations in terms of organs being affected, age of onset and the rate of progression. With such variability, the diagnosis becomes even more challenging. Therefore, mutational analysis along with accurate heteroplasmy detection in the mtDNA is an important part of the diagnosis. Thus, there is need for accurate and faster mutation detection methods for patients that are suspected to carry a mitochondrial disease.

The current molecular diagnostic methods for the detection of mtDNA mutations involves several different and complimentary methods. The detection of mutations is approached by first screening for a panel of point mutations that have been commonly associated with the mitochondrial diseases, followed by the quantification of the mutant load. In case none of the point mutations show up in the screening, the whole genome sequencing of mtDNA is performed to identify rare or novel mutations that might be associated with the disease. Also, in order to analyze large deletions within the genome, a an additional step of Southern blotting needs to be performed. Zhang et al, however, developed a novel approach to analyze the mtDNA in “single” step.

The method employed for the 1-step technique is to first enrich the entire mtDNA using amplification by PCR followed by massively parallel sequencing to detect point mutations as well as large heteroplasmic deletions simultaneously. A total of 45 samples were analyzed for the evaluation of analytic sensitivity and specificity. As stated by the authors “Our analysis demonstrated 100% diagnostic sensitivity and specificity of base calls compared to the results from Sanger sequencing” and added ” the method also detected large deletions with the breakpoints mapped”. Apart from the fact that the 1-step technique is less complex, the detection of point mutations has been found to be more accurate compared to Sanger sequencing that doesn’t provide any quantitative information and falls short of detecting heteroplasmy lying below 15%.

Thus,  the 1-step technique developed by Zhang et al has been demonstrated to be better than the combination of methods currently utilized for the detection of mtDNA mutations in terms of simplicity, cost effectiveness and accuracy.

Sources:

http://www.ncbi.nlm.nih.gov/pubmed?term=Comprehensive%201-Step%20Molecular%20Analyses%20of%20Mitochondrial%20Genome%20by%20Massively%20Parallel%20Sequencing

https://pharmaceuticalintelligence.com/2012/07/09/mitochondria-more-than-just-the-powerhouse-of-the-cell/

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