Polymerase Chain Reaction (PCR): Steps in This DNA Test (original) (raw)

• What is PCR (polymerase chain reaction)?
• How is PCR (polymerase chain reaction) done?
• What is the purpose of doing a PCR (polymerase chain reaction)?
• How was PCR (polymerase chain reaction) discovered?
• What is RT PCR?

What is PCR (polymerase chain reaction)?

PCR (polymerase chain reaction) is a method to analyze a short sequence of DNA (or RNA) even in samples containing only minute quantities of DNA or RNA. PCR is used to reproduce (amplify) selected sections of DNA or RNA. Previously, amplification of DNA involved cloning the segments of interest into vectors for expression in bacteria, and took weeks. But now, with PCR done in test tubes, it takes only a few hours. PCR is highly efficient in that untold numbers of copies can be made of the DNA. Moreover, PCR uses the same molecules that nature uses for copying DNA:

• Two "primers", short single-stranded DNA sequences that are synthesized to correspond to the beginning and ending of the DNA stretch to be copied;
• An enzyme called polymerase that moves 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 (polymerase chain reaction) 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, a device 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 (201.2 F), the double-stranded DNA melts and opens into two pieces of single-stranded DNA.
2. Annealing: At medium temperatures, around 54 C (129.2 F), the primers pair up (anneal) with the single-stranded "template" (The template is the sequence of DNA to be copied.) On the small length of double-stranded DNA (the joined primer and template), the polymerase attaches and starts copying the template.
3. Extension: At 72 C (161.6 F), the polymerase works best, and DNA building blocks complementary to the template are coupled to the primer, making a double stranded DNA molecule.

With one cycle, a single segment of double-stranded DNA template is amplified into two separate pieces of double-stranded DNA. These two pieces are then available for amplification in the next cycle. As the cycles are repeated, more and more copies are generated and the number of copies of the template is increased exponentially.

What is the purpose of doing a PCR (polymerase chain reaction)?

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, establish paternity or biological relationships, etc.. Accordingly, PCR has become an essential tool for biologists, DNA forensics labs, and many other laboratories that study genetic material.

How was PCR (polymerase chain reaction) 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 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:

• The synthesis of cDNA (complementary DNA) from RNA by reverse transcription (RT) and
• The amplification of a specific cDNA by the polymerase chain reaction (PCR).

RT-PCR has been used to measure viral load with HIV and may also be used with other RNA viruses such as measles and mumps.

Previous contributing author and editor:
Medical Author: Frederick Hecht, MD, FAAP, FACMG
Medical Editor: Leslie J. Schoenfield, M.D., Ph.D.

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References

"Tools for genetics and genomics: Polymerase chain reaction"
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