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DNA Amplification for Forensic Analysts

Deoxynucleotide Triphosphates (dNTPs)

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The four dNTPs should be used at equivalent concentrations to minimize misincorporation errors. Lower dNTP concentrations minimize the mispairing at non-target sites and reduce the likelihood of extending misincorporated nucleotides.

Misincorporated bases cannot be proofread since Taq lacks a 3' to 5' exonuclease activity and mismatched bases are inefficiently extended. Misincorporation errors that do occur during PCR promote chain termination. Chain termination restricts the amplification of defective molecules and helps to maintain fidelity.

Stock dNTP solutions are generally neutralized to a pH 7.0. The stability of dNTPs during repeated cycles of PCR is such that approximately 50% of the dNTPs remain after 50 cycles.11 The concentration is usually between 20 and 200µM each.

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