Method 2-PCR

 

A problem encountered in forensics is the attainment of an adequate sample for DNA extraction. A sample may be small or contaminated yielding insufficient DNA for analysis. The introduction of PCR by Kary Mullis in 1985 enabled the amplification of small amounts of DNA.

   

The theory behind PCR is based on certain aspects of DNA replication. An enzyme known as DNA polymerase is used which functions by helping to expand a short sequence into a longer one or a polymer. The enzyme requires single stranded DNA to act as a template for the synthesis of a new strand. Double stranded DNA is heated which will cause the strands to dissociate and therefore become single stranded. Cooling the reaction will cause the strands to reassociate or anneal. DNA polymerase also requires a small portion of double stranded DNA known as a primer to initiate synthesis.

 

Each separated strand can serve as a template providing there is a primer available to each. To cause the primers to bind the reaction is cooled. The primers are chosen to flank the DNA region to be amplified with new primer binding sites being generated on each strand synthesized. The reaction is reheated and the original and synthesized strands separated. The reaction is re-cooled, primers rebind and DNA is synthesized. The net consequence of several cycles of PCR is amplification of the specified region.

 

When first introduced this reaction had to be warmed and cooled manually. The process is now fully automated and the time and temperature can be programmed for repetitive cycles of heating and cooling. The polymerase used is Taq polymerase from bacteria living in hot springs.

 

 

Each cycle is repetitious and is summarized in the following way:

Step 1 Separate strands.

Step 2 Anneal primers.

Step 3 Extend new chain with Taq polymerase

 

Forensic samples are often contaminated making the extraction of DNA difficult. In addition, cross contamination between DNA specimens or from previous reactions can alter results. It is also necessary to be aware of the area of DNA to analyze and how to choose appropriate primers. The reaction is highly sensitive, requiring specific conditions and small concentrations, therefore attention to detail is essential, especially when conducted in the forensic setting.

 

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