DNA TYPING OF FORENSIC MATERIAL WITH MIXED GENOTYPES USING ALLELE-SPECIFIC ENZYMATIC AMPLIFICATION (POLYMERASE CHAIN-REACTION)

Biological material in forensic casework frequently contains a mixture of genotypes, with a predominance of material from the victim and only trace amounts from the person committing the crime. Physical separation of the two genotypes or preferential lysis of different cell types may sometimes be possible. However, it is often difficult to achieve complete separation due to the lysis of cells or lack of material. We have developed an enzymatic amplification system for the HLA DQA1 locus, that will allow the presence of individual alleles in a sample with mixed genotypes to be determined, independent of their initial proportion in the sample. This system permits the identification of an allele representing less than 10(-4) of the background genotype. Use of polymerase chain reaction (PCR) with general primers allows only alleles representing more than about 1% to be detected, while the allele-specific amplification represents up to a 1000-fold increase in sensitivity. This method was applied to a rape case and a combined rape and murder case; in both cases the biological evidential materials contained a mixture of alleles from the victim and the rapist. Allele-specific PCR revealed the presence of alleles identical to those of the suspect using DNA from a vaginal swab taken after a rape incident, whereas by using general primers in the PCR only trace amounts of alleles other than those of the victim were found. Similarly, allele-specific amplification of DNA from vaginal swabs from the murder case revealed the presence of alleles identical to those of the suspect, while standard PCR only indicated the presence of genetic material from the victim.