A rapid chemiluminescent method for quanititation of human DNA

Abstract

A sensitive and simple method for the quantitation of human DNA is described. This method is based on probe hybridization to a human alpha satellite locus, D17Z1. The biotinylated probe is hybridized to sample DNA immobilized on nylon membrane. The subsequent binding of streptavidin-horseradish peroxidase to the bound probe allows for chemiluminescent detection using a luminol-based reagent and X-ray film. Less than 150 pg of human DNA can easily be detected with a 15 minute exposure. The entire procedure can be performed in 1.5 hours. Microgram quantities of nonhuman DNA have been tested and the results indicate very high specificity for human DNA. The data on film can be scanned into a computer and a commercially available program can be used to create a standard curve where DNA quantity is plotted against the mean density of each slot blot signal. The methods described can also be applied to the very sensitive determination of quantity and quality (size) of DNA on Southern blots. The high sensitivity of this quantitation method requires the consumption of only a fraction of sample for analysis. Determination of DNA quantity is necessary for RFLP and many PCR-based tests where optimal results are obtained only with a relatively narrow range of DNA quantities. The specificity of this quantitation method for human DNA will be useful for the analysis of samples that may also contain bacterial or other non-human DNA, for example forensic evidence samples, ancient DNA samples, or clinical samples.

An investigation of the rigor of interpretation rules for STRs derived from less than 100 pg of DNA

“…The DNA was extracted from semen using the Chelex extraction method [21] and quantification was carried out as described by Walsh et al [22]. DNA dilutions were prepared from the stock DNA using sterile distilled water (SDW).…”

DNA typing of fingerprints using capillary electrophoresis: Effect of dactyloscopic powders

“…This proElectrophoresis 1999, 20, 2870±2876 DNA typing of fingerprints 2871 cedure is based on the hybridization of a biotinylated oligonucleotide probe to DNA samples immobilized on a nylon membrane [15]. The probe used is complementary to a specific alpha satellite DNA sequence at the locus D17Z1 [16].…”

Further Development and Validation of DNA-Based Methods for Species of Origin Determination and Human DNA Quantitation in Forensic Casework Specimens

“…The quantity of human DNA is estimated by judging the intensity of the standards compared with that of the specimens. This evaluation is subjective, and there may be variation among analysts or laboratories (3,4). The analysis can also be done using a CCDBIO (MiraiBio) imaging system.…”