• What is PCR (Polymerase Chain Reaction)?
• How is PCR done?
• What is the purpose of doing a PCR?
• How was PCR discovered?
• What is HCV PCR?
• What is RT PCR?

What is PCR (Polymerase Chain Reaction)?

PCR (Polymerase Chain Reaction) is a key technique in molecular genetics that permits the analysis of any short sequence of DNA (or RNA) without having to clone it. PCR is used to reproduce (amplify) selected sections of DNA. Previously, amplifying was done in bacteria, and took weeks. But now, with PCR done in test tubes, it takes only a few hours. PCR is highly efficient so that untold numbers of copies can be made of the DNA. What is more, PCR uses the same molecules that nature uses for copying DNA:

• Two "primers" that flag the beginning and end of the DNA stretch to be copied;
• An enzyme called polymerase that walks along the segment of DNA, reading its code and assembling a copy; and
• A pile of DNA building blocks that the polymerase needs to make that copy.

How is PCR done?

As illustrated in the animated picture of PCR, three major steps are involved in a PCR. These three steps are repeated for 30 or 40 cycles. The cycles are done on an automated cycler, which rapidly heats and cools the test tubes containing the reaction mixture. Each step -- denatauration (alteration of structure), annealing (joining), and extension -- takes place at a different temperature:

1. Denaturation: At 94°C, the double-stranded DNA melts and opens into single-stranded DNA.
2. Annealing: At 54°C, hydrogen bonds form and break between the single-stranded "primer" and the single-stranded "template." (The template provides the pattern to be copied.) The more stable bonds last longer and on that little length of double-stranded DNA (the joined primer and template), the polymerase attaches and starts copying the template.
3. Extension: At 72°C, the polymerase works best. As a result, the attraction, created by the hydrogen bonds, of the primers to the template is stronger than the forces breaking these attractions. The upshot is that bases complementary to the template are coupled to the primer.

What is the purpose of doing a PCR?

To do PCR, the original DNA that one wishes to copy need not be pure or abundant. It can be pure but it also can be a minute part of a mixture of materials. So, PCR has found widespread and innumerable uses -- to diagnose genetic diseases, do DNA fingerprinting, find bacteria and viruses, study human evolution, clone the DNA of an Egyptian mummy, etc.. Accordingly, PCR has become an essential tool for biologists, DNA forensics labs, and many other laboratories that study genetic material.

How was PCR discovered?

PCR was invented by Kary Mullis. At the time he thought up PCR in 1983, Mullis was working in Emeryville, California for Cetus, one of the first biotechnology companies. There, he was charged with making short chains of DNA for other scientists. Mullis has written that he conceived of PCR while cruising along the Pacific Coast Highway 128 one night on his motorcycle. He was playing in his mind with a new way of analyzing changes (mutations) in DNA when he realized that he had instead invented a method of amplifying any DNA region. Mullis has said that before his motorcycle trip was over, he was already savoring the prospects of a Nobel Prize. He shared the Nobel Prize in chemistry with Michael Smith in 1993.

As Mullis has written in the Scientific American: "Beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar molecules in an afternoon. The reaction is easy to execute. It requires no more than a test tube, a few simple reagents, and a source of heat."

What is HCV PCR?

HCV PCR (polymerase chain reaction) is a test for the hepatitis C virus ( HCV ). There are three types of HCV PCR tests:

1. The HCV PCR viral detection test: This qualitative test is designed to detect whether the hepatitis C virus is or is not present.
   
   
2. The HCV PCR viral load test: This quantitative test looks for the virus and estimates the number of HCV viruses per ml of blood.
   
   
3. The HCV PCR genotype test: This test looks for the virus and determines the particular subtype of HCV.

The three different HCV PCR tests serve different purposes. The qualitative test is done to confirm the presence of HCV in the blood of a person with positive HCV antibody test. The quantitative test is done to estimate the length of treatment that will be needed and to monitor the effectiveness of that treatment. The genotype test is usually done before the start of treatment because it can differentiate each of the major subtypes of HCV. Knowing the subtype can be important because, for example, treatment with interferon is more often effective for people with genotype subtypes 2 or 3 than for those with genotype 1.

What is RT PCR?

RT-PCR (Reverse transcriptase-polymerase chain reaction) is a highly sensitive technique for the detection and quantitation of mRNA (messenger RNA). The technique consists of two parts:

1) The synthesis of cDNA (complementary DNA) from RNA by reverse transcription (RT) and

2) The amplification of a specific cDNA by the polymerase chain reaction (PCR).

RT-PCR has been approved by the FDA as a test to measure viral load with HIV . It may also be used with other RNA viruses such as measles , mumps , and metapneumovirus.

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