Glossary

PCR: Polymerase Chain Reaction

Polymerases (DNA): is an enzyme that builds a new strand of DNA

DNA:   Deoxyribonucleic Acid

Amplify:  an increase in the number of copies of a piece of DNA.

Oligonucleotide:  (oligo means few) a short length of DNA nucleotides (2-50).

Nucleotide:  a subunit or base of DNA made up of adenine (A), guanine (G), thymine (T), or cytosine (C).

Primer:  a short stretch of DNA (oligonucleotide) needed to start the DNA polymerase reaction

Polymerase Chain Reaction (PCR)

PCR: a method for specifically amplifying a region of DNA

Requirements:

PCR is a process based on a specialized polymerase enzyme, which can synthesize a complementary strand to a given DNA strand in a mixture containing the 4 DNA bases and 2  DNA fragments (primers, each about 20 bases long) flanking the target sequence.  The mixture is heated to separate the strands of double-stranded DNA containing the target sequence and then cooled to allow (1) the primers to find and bind to their complementary sequences on the separated strands and (2) the polymerase to extend the primers into new complementary strands.  Repeated heating and cooling cycles multiply the target DNA exponentially, since each new double strand separates to become two templates for further synthesis.  In about 1 hour, 20 PCR cycles can amplify the target by a million-fold.

The PCR provides an extremely sensitive means of amplifying small quantities of DNA. The development of this technique resulted in an explosion of new techniques in molecular biology (and a Nobel Prize for Kary Mullins in 1993) as more and more applications of the method were published. The technique was made possible by the discovery of Taq polymerase, the DNA polymerase that is used by the bacterium Thermus auquaticus that was discovered in hot springs. This DNA polymerase is stable at the high temperatures need to perform the amplification, whereas other DNA polymerases become denatured (destroyed).

The cycling reactions :
There are three major steps in a PCR, which are repeated for 30 or 40 cycles. This is done on an automated cycler, which can heat and cool the tubes with the reaction mixture in a very short time.

Denaturation at 94°C
During the denaturation, the double strand melts open to single stranded DNA, all enzymatic reactions stop (for example: the extension from a previous cycle).

Annealing at 55°C
The primers are bouncing around, caused by the Brownian motion. Hydrogen bonds are constantly formed and broken between the single stranded primer and the single stranded template. The more stable bonds last a little bit longer (primers that fit exactly) and on that little piece of double stranded DNA (template and primer), the polymerase can attach and starts copying the template. Once there are a few bases built in, the hydrogen bond is so strong between the template and the primer, that it does not break anymore.

Extension at 72°C
This is the ideal working temperature for the polymerase. The primers, where there are a few bases built in, have a stronger attraction to the template, created by hydrogen bonds, than the forces breaking these attractions. Primers that are on positions with no exact match, get loose again (because of the higher temperature) and don't give an extension of the fragment.  The bases (complementary to the template) are coupled to the primer on the 3' side (the polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template)

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