BACKGROUND: With the discovery of DNA polymerase in 1956, an enzyme that replicates DNA, a new world of science was opened. The next big discovery in 1970 was Reverse Transcriptase (RT) that uses RNA as a template to synthesize DNA. Later, DNA polymerase1 was discovered in microorganisms living at high temperature in the walls of a volcano and the enzyme was thermally stable (Taq Polymerase). Polymerase Chain Reaction (PCR) was developed, using these enzymes. In 1985  refinements led to QPCR (Quantitative PCR for DNA) and QRT-PCR (for RNA) and demonstrated convenient quantitation. These advancements drastically improved diagnostic medicine and created new areas of molecular biology where genes from a single cell can be analyzed and low levels of microbes and virus particles are detected. For a good explanation of how PCR and Q-PCR works please click on the link to our web page marinbio.com/PCR-QPCR.

WHY USE QPCR AND PCR: There are many diverse applications and benefits of PCR and QPCR of which a few are described below.

COV-2 VIRUS DETECTION and OTHER RNA VIRUS’S: The most current topical application of QPCR is the detection of the COV-2 virus. The RNA genes of the virus are transcribed into DNA and detected by PCR. Because QPCR is relatively quick, results can be obtained within 2 hours for some tests. The primers must be sensitive enough to match the target virus yet specific enough to exclude all others. The many laboratories developing a virus test use different RNA gene sequences.  False positives could stem from the specificity of the DNA primers that initiate the detection reaction, reagents and test methods used, as well as sample collection (right anatomical site and time of infection). So QPCR uses the single -stranded RNA genome from the virus and individual genes can be detected, such as structural proteins, including envelope glycoproteins, the spike protein (seen in the graphic illustration of COV-2), transmembrane proteins, helicase (which unwinds the RNA), the nucleocapsid (the protein shell wrapped around the RNA), and the viral-specific replication genes.  The CDC In the United States recommends at least detection of two nucleocapsid protein targets. To avoid potential cross -reaction with other endemic coronaviruses as well as potential genetic drift of SARS -CoV -2, at least two molecular targets should be included in the assay. All tests should be FDA approved and validated and performed by a qualified laboratory.

MEDICINE: PCR and QPCR enable medicine to fast forward for diagnosis and therapy. Genetic disorders caused by mutations in the DNA are detected by PCR.  The PCR is able to focus on selected segments of DNA, identify the changed in the DNA sequence, resulting in monitoring and potentially customizing the therapy to ameliorate the symptoms. Gene Therapy is moving forward to treat the underlying causes. Genotyping of embryos allows for detection of chromosomal and genetic disorders.  Personalized medicine makes use of data that can allow for therapy (e.g. proper food selection for inflammatory bowel disease). Identification of pathogenic microorganisms, bacteria, virus, parasite, fungi, allows for targeted treatment. Development of new pharmaceuticals employs PCR and QPCR for monitoring gene expression changes in identifying the mechanism of action of potential drugs. Organ transplant relies on PCR, QPCR for improved accuracy of tissue typing, reducing the advent of tissue rejection.

FORENSIC SCIENCE:  The identification of criminals and avoidance of indicting the wrong person enhances our crime prevention. The use of PCR and QPCR to provide evidence collected at the crime scene, such as blood, hair, soil and semen, has been accepted in courtrooms for over 30 years. DNA screening can be used in paternity cases. The analysis of DNA is slightly different than that with viruses and in medicine applications.  DNA contains regions in which short sequences of bases are repeated multiple times in many loci throughout the genome. The exact number of repeats (called short tandem repeats (STRs), at any particular locus varies from person to person, and can be used as markers to identify individuals.  Other markers such as genes for brown hair are also used.

ENVIRONMENTAL AND GMO ANALYSIS:  QPCR is the method of choice to quantitatively measure amounts of transgene DNA (from a different organism) in  a food or feed sample and the number of its copies. For technical reasons, certain DNA sequences are shared by several GMOs, allowing the rapid identification of the foreign DNA in the sample.  The GOOD thing about GMOs, is that allergens can be genetically engineered from foods like peanuts to decrease their adverse effects on consumers, or a more balanced amino acid composition can be added to a “deficient” food. Cross pollination using the traditional and conventional breeding method (brush the pollen from one plant to a new plant), is in fact making GMOs. The EU-funded GMO research and the National Academy of Sciences both have released statements that “genetic engineering is one of the newer technologies available to produce desired traits in plants and animals used for food, and it poses no health risks that cannot also arise from conventional breeding and other methods used to create new foods.”

THIS IS WHAT MarinBio CAN DO:  MarinBio scientists are experts in molecular biology and have used PCR and QPCR for many year. Our scientists have unequivocally shown no GMOs in corn used for corn chips, allowing our client to export the chips to countries with GMO restrictions. We have performed QPCR for our clients identifying molecular mechanisms of action of their drugs which was presented to the FDA.  MarinBio scientists have over 25 years of experience helping our clients to develop, qualify and validate viral or cell-based assays, different RNA/DNA tests and proteins involved in viral infectivity, anti-inflammatory drug potency immunoassays or other quantitative bioassays.  For further information please contact MarinBio at info@marinbio.com or call 415 883 8000 or visit our website at www.marinbio.com.