DNA damage reduces Taq DNA polymerase fidelity and PCR amplification efficiency

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• DNA damage impedes DNA polymerase function, leading to errors.
• Taq DNA polymerase is widely used in PCR but its fidelity with damaged templates is not fully understood.

Purpose of the Study:

• To investigate the impact of specific DNA lesions on Taq DNA polymerase fidelity and PCR amplification efficiency.
• To introduce and validate a new method for quantifying PCR amplification efficiency in damaged DNA templates.

Main Methods:

• Assessed Taq DNA polymerase incorporation errors in the presence of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxodA), and abasic lesions.
• Introduced and utilized the mean modified efficiency (MME) to measure PCR amplification efficiency of damaged DNA templates.

Main Results:

• 8-oxodG caused Taq polymerase to insert dCMP and dAMP.
• 8-oxodA induced dTMP incorporation and significant n-1 deletions.
• Abasic lesions resulted in dAMP incorporation and n-1 deletions.
• MME successfully quantified reduced amplification efficiency for templates with 8-oxodG, 8-oxodA, or abasic sites.

Conclusions:

• Specific DNA lesions differentially affect Taq DNA polymerase fidelity and lead to characteristic errors.
• The MME method provides a sensitive approach to quantify PCR amplification efficiency reductions caused by DNA damage.
• MME can be valuable for assessing DNA degradation in environmental or archival samples.