1. You know that PCR uses heat resistant enzymes. We used Taq polymerase, but th
ID: 253228 • Letter: 1
Question
1. You know that PCR uses heat resistant enzymes. We used Taq polymerase, but there are a number of heat resistant DNA polymerases that can be used. One big difference that is seen is the fidelity, or proofreading ability. Proofreading ability lowers the number of mistakes that the polymerase makes during the extension step of PCR. Taq does not proofread.
a. Why is Taq’s lack of proofreading ability not important for the purpose of this lab? (1 pt.)
b. Provide an example of where it would be important to use a polymerase that does have proofreading capability. (1 pt.)
Explanation / Answer
Polymerase chain reaction is a technique to amplify the desired DNA sequence. This technique requires a reaction mixture consisting of sample DNA, primers, nucleotides and a DNA polymerase.Generally, enzymes are heat sensitive. As large amount of heat is produced during this process, DNA polymerase used should be heat resistant. So, the enzymes extracted from thermophilic bacteria is used. The frequently used heat resistant polymerases are Taq polymerase, Pfu polymerase, and so on.
The polymerase enzyme is used to incorporate a complementary nucleotide to the existing primer by 5’ to 3’ polymerization activity. Most of the polymerases also have proofreading ability, which means to check whether the incorporated nucleotide is the correct match or mismatch. This is called 3’ to 5’ exonuclease activity.
Taq polymerase is the mostly used polymerase for PCR technique. It is having high efficiency (97%) for its 5’ to 3’ polymerization activity and high tolerance to heat.
a. Due to its higher efficiency and low error rate, it does not alter the entire gene product. So, in general detection for the presence or absence of a product, identifying pathogenic organisms, diagnosis tests, insitu-hybridization using fluorescent nucleotides, multicolor detection of gene expression, and so on, it may not be a drawback for the enzyme used.
b. In some cases, the DNA amplified should be 100% as the sample DNA depending on the requirement such as to detect the point mutations, deletions or insertions, cloning, mutagenesis, protein expression. In these cases, it is important to use the enzyme having proofreading ability.
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