What is PCR Testing?

dna-double-helix-marc-phares-and-photo-researchers

PCR is an acronym for polymerase chain reaction. PCR increases the amount of DNA available from a sample for typing and analysis.

Within the DNA string, some areas are the same for all human beings (conserved, or constant). Other areas tend to vary from person to person (variable, or polymorphic). The variable regions are usually scattered among the conserved regions and are used in DNA testing profiles.

Most organisms naturally copy their DNA in the same way. The PCR mimics this process, except replication takes place in a test tube. When any cell divides, enzymes called polymerases make a copy of all of the DNA in each chromosome. DNA polymerase is also used in PCR to make copies of specific target strands.

DNA is made from four nucleotide bases, represented by the letters A (adenine), C (cytosine), G (guanine), and T (thymine). The A on one strand of DNA always pairs with the T on the opposite strand, and C always pairs with G. The two strands are said to be complementary to each other.

To copy DNA, the polymerase requires two additional components: a supply of the four nucleotide bases and something called a primer. DNA polymerases cannot copy a chain of DNA without a short sequence of nucleotides to “prime” the process, or get it started. So the cell has another enzyme called a primase that actually makes the first few nucleotides of the copy. This portion of DNA is called a primer. Once the primer is made, the polymerase can take over making the rest of the new chain.

There are three major steps in PCR. The first step in this process is to denature (unzip, unwind) the target genetic material in the two DNA chains of the double helix. This is done by heating the material to 90-96oC. As the two strands separate, DNA polymerase makes a copy using each strand as a template.

The primers cannot bind to the DNA strands at such a high temperature, so the vial is cooled to 55oC for the second step in the process. At this temperature, the primers bind or “anneal” to the ends of the DNA strands.

The final step of the reaction is to make a complete copy of the originals. The specific polymerase that is used for this process works best at around 75oC, so the temperature of the vial is raised one more time.

The three steps in the polymerase chain reaction – separation of the strands, annealing the primer to the template, and synthesis of new strands – take less than two minutes. At the end of a cycle, each piece of DNA has been replicated.

The process can be repeated to get more of the targeted DNA; the process can be repeated using the DNA that was replicated in the first procedure. The total amount will double every time.