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.
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