Titration of heparinase for removal of the PCR-inhibitory effect of heparin in DNA samples.
Heparin is a naturally occurring polysaccharide found primarily in the liver, lung and artery walls (White et al. 1968), which is commonly used as an anticoagulant for venal blood samples. Although the inhibitory effect of heparin on the polymerase chain reaction (PCR) and other enzyme-mediated reactions has been noted (Beutler et al. 1990; Izraeli et al. 1991), this is not widely known to field biologists collecting samples which are subsequently used for genetic analysis. The enzyme heparinase (Linker & Hovingh 1972) has been used successfully to eliminate heparin inhibition from DNA samples post-extraction, because the normal range of DNA extraction techniques fail to do so (Beutler et al. 1990; Oberhauser et al. 1994; Taylor et al. 1994). However, the cost of heparinase treatment for large numbers of samples, such as those from a population survey, would prove prohibitively expensive if the concentration and grade of heparinase (heparinase II) used by Beutler et al. (1990) is followed (see Table 1): heparinase II is more than three times the price per unit than heparinase I. If the blood-to-heparin ratio is relatively high during the original collection, it appears that the inhibitory effect of heparin can be diluted out. In addition DNA extracted from washed lymphocytes rather than whole blood may not show the same level of inhibition. For example, the DNA extracted from washed lymphocyte pellets from 10 mL heparinized mammalian blood samples is amplifiable in our laboratory. However, DNA extracts from small (less than 1 mL), frozen brushtail possum Trichosurus vulpecula blood samples collected into heparin fail to amplify in PCR reactions. The DNA was extracted by lysis of the whole blood (method in Taylor et al. 1991) in order to reduce the number of decanting steps in the procedure, because yields were expected to be low. I therefore carried out an experiment to examine the efficacy of different concentrations and grades of heparinase in the removal of presumed PCR inhibition from these samples.
Taylor
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Cooperative Research Centre for Conservation and Management of Marsupials, School of Biological Sciences, Macquarie University, Sydney, NSW, Australia. ataylor@rna.bio.mq.edu.au
D000818:Animals; D001483:Base Sequence; D001800:Blood Specimen Collection; D004247:DNA / Q000097:blood* / Q000235:genetics* / Q000302:isolation & purification; D017931:DNA Primers / Q000235:genetics; D006494:Heparin Antagonists; D019763:Heparin Lyase; D009893:Opossums / Q000097:blood / Q000235:genetics; D016133:Polymerase Chain Reaction; D011133:Polysaccharide-Lyases
D017931:DNA Primers; D006494:Heparin Antagonists; D004247:DNA; D011133:Polysaccharide-Lyases; D019763:Heparin Lyase