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.
We have developed a rapid, immobilized probe-based assay for the detection of sequence variation in the hyper-variable segment II (HVII) of the mitochondrial DNA (mtDNA) control region. Using a panel of 17 sequence-specific oligonucleotide (SSO) probes immobilized on nylon membrane strips, we typed 689 individuals from four population groups. The genetic diversity value for each population was calculated from the frequency data, and the frequencies of distinct “mitotypes” in each group were determined. We performed DNA sequence analysis of 129 samples to characterize the sequences associated with “blanks” (absence of probe signals) and weak probe signals. Out of 689 samples, we observed five heteroplasmic samples (excluding the variable C-stretch beginning at position 303) using the immobilized SSO probe panel. The SSO probe strips were used for the analysis of shed hairs and bloodstains from several criminal cases in Sweden, one of which is described here. We conclude that this mtDNA typing system is useful for human identification and significantly decreases casework turnaround time.
Determining the gender of an evidentiary sample can be an important part of casework analyses. Gender information, particularly when combined with mitochondrial DNA analysis, can serve to distinguish biological evidence from two people who share the same DNA type(s) but differ by sex. When typing sexual assault evidence, gender information can serve as confirmation that the “sperm fraction” extracted from swabs and stains actually contains male DNA and also as an indicator of the amount of male DNA present in the non-sperm fraction. The PCR-based assay described here relies on amplification of a small, polymorphic region of a homologous zinc finger protein locus present on the X and Y chromosomes. The gender of the sample donor is determined from the PCR product either by Haelll restriction enzyme digestion followed by gel electrophoresis or by hybridization to immobilized sequence specific oligonucleotide probes (reverse dot blot). When using the reverse dot blot approach, amplification and typing of the gender PCR product can be coupled to amplification and typing of the AmpliType® HLA DQα and PM markers. Sensitivity and mixture studies were performed in addition to the analysis of casework bloodstains and sexual assault kit samples. Additional studies using this gender determination assay are described in the accompanying paper.
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