Journal Published Online: 01 March 1994
Volume 39, Issue 2

Identification of the Heme Compound Copurified with Deoxyribonucleic Acid (DNA) from Bloodstains, a Major Inhibitor of Polymerase Chain Reaction (PCR) Amplification

CODEN: JFSCAS

Abstract

The heme compound found in deoxyribonucleic acid (DNA) extracted from bloodstains, which is regarded as a major inhibitor of polymerase chain reaction (PCR), was characterized in comparison with alkaline and acid hematin, histidine and ammonia hemochromogens, and globin and serum albumin hemochromogens digested by proteinase K. Alkaline and acid hematin were almost completely removed by phenol/chloroform treatment and ethanol precipitation, so as not to be copurified with DNA from the specimens. Spectrophotometric results indicated that the contaminant was likely to be the product of proteinase K digestion of some heme-blood protein complex, which was not completely extracted by organic solvents and remained in the ethanol precipitates of DNA. The results of polyacrylamide gradient gel electrophoresis and intensity of the inhibition of PCR suggested that the ligand of the contaminant was a somewhat large molecule, resistant to the proteolysis by proteinase K. The addition of bovine serum albumin to the reaction mixture prevented the inhibition of PCR by the heme compounds, probably by binding to the heme. This showed that the inhibition was not due to the irreversible inactivation of the enzyme.

Author Information

Akane, A
Kansai Medical University, Moriguchi, Osaka, Japan
Matsubara, K
Shimane Medical University, Izumo, Japan
Nakamura, H
Shimane Medical University, Izumo, Japan Criminal Investigation Laboratory, Shimane Prefectural Police Headquarters, Matsue, Japan
Takahashi, S
Shimane Medical University, Izumo, Japan
Kimura, K
Shimane Medical University, Izumo, Japan
Pages: 11
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Stock #: JFS13607J
ISSN: 0022-1198
DOI: 10.1520/JFS13607J