PCR

PCR is a technique used to amplify specific regions of a DNA molecule. In order to understand what happens during PCR it is necessary to review a few important characteristics of the DNA molecule.

• DNA is composed of two strands
• Hydrogen bonds hold the two strands together
• Base pairing in the two strands is complementary
  • A-T pairs form 2 hydrogen bonds
  • G-C pairs form 3 hydrogen bonds
• The two strand are antiparallel.
  • 5' end has a PO₄ group
  • 3' end has a OH group
• Direction of replication is 5' to 3' in each strand

Sometimes during replication errors occur and the DNA polymerase slips and inserts extra copies of some of the bases. When this happens tandem repeats can occur. Consider a simple situation where the ploymerase slips when inserting the CG sequence and an extra tandem pair is inserted. If the error occurs randomly over many generations the number of CG sequences in the DNA molecule could become quite variable from individual to individual. Regions of this type are known as Variable Nucleotide Tandem Repeat sequences (VNTR). VNTRs are present throughoutt the human genome and because of the unique combination of VNTRs they are as good as fingerprints at identifying individuals.

How is this possible. Let's examine a simple situation using CG repeats as an example. Consider two individuals. The CG pattern of these individuals is clearly different, but how can you detect the difference? This is where PCR comes in.

PCR

Begin with a DNA template - double stranded DNA for which the sequence of bases on either side of the region of interest is known

Select primers- single stranded DNA used to prime the DNA polymerase

• two are used
• bracket region of interest
• complement opposite strands of the DNA template
• 3' ends face towards the region to be copied

Begin PCR

1. Add the DNA template to a mixture containg the necessary ingredients:

• dNTP - subunits of DNA composed of sugar, phosphate and one of four bases (A, T, G, C)
• DNA polymerase - taq polymerase is not sensitive to heat
• Mg²⁺ - a metal ion required for DNA polymerase activity
• appropriate primer sequence

2. Heat the mixture to 94^oC to break the hydrogen bonds between the strands of DNA.

3. Cool the mixture 60^oC to 72^oC to allow 5' ends of the primers to bind at the 3' ends of the single DNA strands.

4. Allow time for the taq polymerase to move down the single strands adding the appropriate dNTPs and creating double stranded copies.

Alternate steps 2-4 35x to produce over a billion copies (2³⁵) of the template.

This process allows very small quantities of DNA to be replicated provided the appropriate primers are available. If a large sample of DNA is contaminated with a small amount of DNA from a second individual copies of the contaminating DNA will also be replicated but will be less abundant in the final sample. However if you begin with small samples then the contaminant cannot be distinguished. Care must be used in this process.

To view an animation of the PCR process go to DNAi.org/b/index.html. Once there select TECHNIQUES at the bottom of the page and then "amplification" at the top of the next page. Select PCR animation to view the process. While at this site you may also want to explore electrophoresis under the "sorting and sequencing" tab. This DNA site was funded by Howard Hughes Medical Institute and has many other interesting facts about DNA to explore. Use the back button to return to this page.

Questions