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A Study of PCR Inhibition Mechanisms Using Real Time PCR

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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.

Keywords: DNA typing; HUMTH01; calcium; forensic science; hematin; humic acid; inhibition; melanin; real time polymerase chain reaction

Document Type: Research Article


Affiliations: 1: Division of Science and Mathematics, Upper Iowa University, PO Box 1857, Fayette, IA 52142. 2: Center for Neurological Diseases, Brigham & Women’s Hospital, Harvard Institutes of Medicine, 77 Avenue Louis Pasteur Room 785, Boston, MA 02115-5817. 3: Department of Chemistry and Biochemistry, Florida International University, CP304, 11200 SW 8th Street, Miami, FL 33199.

Publication date: January 1, 2010

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