Polymerase Chain Reaction (PCR) - Paternity Test - USMLE

What is PCR?
PCR stands for Polymerase Chain Reaction. It is a technique used to amplify a targeted segment of DNA into million-folds within few hours by using a heat-stable enzyme called DNA polymerase, hence the name of the technique.

What are the applications of PCR?

1. Forensic analysis of a trace amount of DNA
2. Genetic fingerprinting
3. Diagnosing malignant diseases like leukemia and lymphoma
4. Paternity testing
5. Direct mutation testing
6. Bacterial and viral infections diagnosis, especially those which are uncultivable or slow-growing
7. HIV testing
8. To measure viral load in patients with AIDS

Describe the procedure of PCR.

• Identify the target sequence first in the DNA segment that requires amplification
• Once identified, add the DNA segment containing the target sequence into the sample
• Add primers that are complementary to both 3` flanks of the target sequence for amplification
• Next, add Taq DNA polymerase, which is a heat-stable enzyme.
• Add dNTPs (deoxyribonucleotides) required for DNA synthesis
• Apply heat to denatured DNA and resulting in dsDNA into ssDNA
• Cool the sample to allow annealing of DNA.
• Now, heat the sample again to increase Taq DNA polymerase activity in order to allow primer elongation and new complementary strands synthesis.
• Process to be repeated for ~20 cycles. At the end, million-folds amplification of target sequence would have been achieved.

  Note: Always add excess primers so that adequate amount of primers are available to make the ratio of primers to complementary strands high enough to allow for complementary binding.

Now to test your knowledge: USMLE - styled question:



Answer: Suspected Father 2 is the biological father of child.

Explanation: We know that mother is biologically related to the child. Now in order to determine who is the biological father of the child, we must compare child’s DNA segment with that of the other two potentially suspected fathers. In order to do so, we must first collect a segment of DNA each from the mother, child and the two suspected fathers.

Now after applying PCR-amplified DNA by electrophoresis on agarose gel, we come up with the picture shown above.

We need to look for the matching combination between the mother and the other men and see whether they match with that of the child’s. It is important that the child’s DNA target sequence must be a combination of mother’s and the potential’s father’s DNA sequences. The only possible suspect is Father No. 2