It is not certain what the role of Chelex is in DNA preparation for PCR. When we have subjected purified DNA to the boiling treatment in these procedures with Chelex, or else in distilled water alone, the DNA treated by boiling without Chelex becomes inactive in PCR. If the DNA is boiled in 0.0 1 M Tris-HCI. pH 8.0 with 0.1 M EDTA, it is also protected for PCR. This is in agreement with the protective role for Chelex that Singer-Sam et al. (11)suggest in which Chelex sequesters divalent heavy metals that would otherwise introduce DNA damage. A model for the extraction process is that the boiling treatment is useful for releasing DNA from low numbers of cells, and that Chelex protects the DNA from the effects of the boiling. Note that we have found that, unlike other cells, sperm do not release DNA from the boiling treatment alone; pretreatment with proteinase K and DTT treatment is required. Chelex can chelate a large amount of divalent ions that may be donated by the sample, and the Chelex beads can be easily removed so that they will not interfere with subsequent PCR amplifications that require Mg++.
An additional motivation for our testing alternative sample preparation methods is that we have found that DNA prepared from bloodstains sometimes contains inhibitors of PCR (6; E. Blake, personal communication). We have found that extractions of bloodstains prepared by the Chelex-based procedure are less likely to have inhibitors of PCR than extracts prepared by proteinase treatment and phenol-chloroform extraction. For example, two of the CACLD bloodstain samples initially showed inhibition of PCR when extracted using conventional phenol-chloroform methods, but no inhibition was observed when these samples were extracted using the Chelex methods. Previous work (6) has suggested that porphyrin compounds from blood are the cause of the inhibition.
Because no proteinase treatment is used that would release heme from globin, it is possible that less free porphyrin compounds are in the Chelexprcpared extracts. Also, it is possible that such inhibitors are bind ing to the Chelex bead matrix itself.
In summary, we have presented procedures that should permit the rapid preparation of DNA from forensic-type samples. Since far fewer manipulations are required, these procedures should also reduce the chance of inadvertent contamination of samples by extraneous DNAs.
We thank C.T. Corney, M. Kuo, J. Maholovich, N. Fildes and E. Blake for providing samples. We thank C. Levenson, L. Goda and D. Spasic for providing oligonucleotides. We thank S. Roe for assistance in formatting protocols. We thank the Forensics Project team at Cetus for support and encouragement.
1.) AmpliType User Guide, Section 3 (Sample Preparation).
2.) Brunk, C.F, K.C. Jones, and T.W. James. 1979. Assay for nanogramquantities of DNA in cellular homogenates. Anal. Biochem. 92:497-500.
3.) Budowle, B., and S. Baechtel. 1990. Modifications to improve the effectiveness of restriction fragment length polymorphism typing. Appl. Theor. Electrophoresis. 1:181-187.
4.) Dykes, D. 1988. The use of biotintylated DNA probes in parentage testing: non-isotopic labeling and non·toxic extraction. Electrophoresis 9:359-368.
5.) Grimberg, J.S, L. Nawoschic, R. Bellvscio, A. McKee, and A. Turck Eisenberg. 1989. A simple and efficent non-organic proceedure for the isolation of genomic DNA from blood. Nucleic Acids Res. 22:8390.
6.) Higuchi, R. 1989.Simple and rapidpreparation of samples for PCR. In H.E. Erlich (Ed.) PCR Technology. Principles and Applications for DNA Amplification Stockton Press, New York:31-38.
7.) Kapuscinski, J, and B. Skocylas. 1977. Simple and rapid fluorimetric methods for DNA microassay. Anal. Biochem. 83:252-251.
8.) Maniatis, T., E.F. Fritsch, and J Sambrook. 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
9.) Oppitz, E. 1969. Eine Neue Farbemthode Nachweis der Spennien be: SittlichkeilSdelit.ten. Arkiv fur Krimin 144:145-148.
10.) Saiki, R, P.S. Walsh, C. Levenson, and H. Erlich. 1989. Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes. Proc. Natl. Acad. Sci. USA 86:6230-6234.
11.) Singer-Sam, J, R.L. Tanguay, and A.O. Rjggs. 1989. Use of Chelex to improve PCR signal from a srnall number of cells. Amplifications: A Forum for PCR Users:11.
12.) U.S. Patent No.4.682,202 and No. 4,683,195 to CetusCorporation.
13.) Vogelstein, B., and D. Gillespie. 1979. Preparation and analytical purification of DNA from agarose. Proc. Natl. Acad. Sci. USA 76:615-619.
14.) Valsh, P.S., N. Fildes, A. Louie, and R. Higuchi. 1991. Report of the blind trial of the Cetus AmpliType HLA DQα DNA amplification and typing kit. J. Forensic Sci..
15.) Watson, R. 1989. The formation of primer artifacts in polymerase chain reactions. Amplifications: A Forum for PCR Users:5.
In order to deliver a personalised, responsive service and to improve the site, we remember and store information about how you use it. This is done using simple text files called cookies which sit on your computer.