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A Study of PCR Inhibition Mechanisms Using Real Time PCR * , †
Author(s) -
Opel Kerry L.,
Chung Denise,
McCord Bruce R.
Publication year - 2010
Publication title -
journal of forensic sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.715
H-Index - 96
eISSN - 1556-4029
pISSN - 0022-1198
DOI - 10.1111/j.1556-4029.2009.01245.x
Subject(s) - amplicon , polymerase chain reaction , primer (cosmetics) , primer dimer , real time polymerase chain reaction , locus (genetics) , melting temperature , microbiology and biotechnology , dna , melting point , hot start pcr , chemistry , biology , gene , genetics , multiplex polymerase chain reaction , materials science , organic chemistry , composite material
In this project, real time polymerase chain reaction (PCR) was utilized to study the mechanism of PCR inhibition through examination of the effect of amplicon length, melting temperature, and sequence. Specifically designed primers with three different amplicon lengths and three different melting temperatures were used to target a single homozygous allele in the HUMTH01 locus. The effect on amplification efficiency for each primer pair was determined by adding different concentrations of various PCR inhibitors to the reaction mixture. The results show that a variety of inhibition mechanisms can occur during the PCR process depending on the type of co‐extracted inhibitor. These include Taq inhibition, DNA template binding, and effects on reaction efficiency. In addition, some inhibitors appear to affect the reaction in more than one manner. Overall we find that amplicon size and melting temperature are important in some inhibition mechanisms and not in others and the key issue in understanding PCR inhibition is determining the identity of the interfering substance.