Question

Polymerase Chain Reaction (PCR) uses a special heat-stable DNA polymerase (Taq polymerase) that is slightly less accurate than DNA polymerase (Pol I) purified from E. coli. Taq polymerase will therefore introduce wrong bases into a growing DNA chain more frequently than will Pol I.

In which one or more of the following applications of PCR will this type of inaccuracy be a problem? Explain why.

1. Detection of bacterial DNA in infected tissues from patients.
2. Detection of viral RNA in infected cells from patients.
3. Amplification of a gene from bacterial genomic DNA in order to clone it into a bacterial vector to determine the sequence.
4. Amplification of a gene from a cDNA library in order to clone it into a bacterial vector for protein expression.

Answer 1

Bacterial DNA can be identified when PCR product of the 16s gene is obtained, sequenced and aligned against database.

PCR uses Taq polymerase which is a heat-tolerant bacterium. Its very heat stable and is most active around 70°C and PCR works at that temperature. DNA polymerase would be non functional at that temperature.

The problem would be for Detection of viral RNA in infected cells from patients. Since we have to identify RNA and not DNA. DNA polymerase adds bases pairs only to dna.