DNA damage promotes mistyping in the allele specific oligonucleotide probing analysis of forensic samples

Abstract

Five polymerase chain reaction (PCR) products which could not be reliably typed by allele-specific oligonucleotide (ASO) probing at the human leukocyte antigen (HLA) DQA1 locus were analyzed by polyacrylamide gel electrophoresis and direct sequencing. The first method revealed the preferential amplification of only one of the two alleles in two cases. Direct sequencing of PCR products allowed unambiguous genetic typing but a high number of artifacts was observed. Several of these artifacts occurred in the sequences recognized by the ASOs. This finding provides an explanation for the mistyping in the ASO probing procedure because Taq polymerase errors both created new genetic specificities and eliminated site-specific polymorphisms. Reversed-phase HPLC-MS of the five forensic templates showed a high degree of DNA damage. These data together indicate that the risk of mistyping when using the ASO probing procedure cannot be neglected in the forensic analysis of damaged DNA samples.

Major histocompatibility complex and microsatellite variation in two populations of wild gorillas

“…We excluded three published sequences (Gogo1*0306, Gogo5*0201, Gogo5*0401) that are probably products of in vitro recombination. This occurrence of artificial sequences while typing the MHC‐DRB has been both described and demonstrated experimentally by others (Fattorini et al . 2000; Longeri et al .…”

“…1998). Concern regarding the low amount of template present in the PCRs led us to use 40 cycles in the amplifications of the MHC‐DRB loci, and the proportion of apparent recombinants we observed was similar to that described from the use of damaged DNA from forensic samples (Fattorini et al . 2000).…”

“…Amplification conditions for all MHC PCRs consisted of an initial denaturation at 95 °C for 3 min, 40 cycles of 90 s at 95 °C, 45 s at 52 °C, and 45 s at 72 °C, followed by 30 min at 72 °C. Following typical practice to avoid spurious results (Fattorini et al 2000;Nino-Vasquez et al 2000), the products of three independent successful amplifications were analysed per individual.…”

Molecular characterisation of the nucleic acids recovered from aged forensic samples

“…Previous studies [19,24] showed a high frequency of sequencing artefacts and unreliable genetic characterisations in aged forensic samples. Identification and quantification of human DNA in the extracts from the biological samples and the degree of chemical decay of its primary structure should be considered for a reliable evaluation of the PCR typing results.…”

Reply: facts, faeces and setting standards for the study of MHC genes using noninvasive samples

“…1999), sequences that arise from nucleotide misincorporations or artefactural recombination in PCRs that start from few or single molecules can only be detected by screening more than one reaction (e.g. Fattorini et al . 2000; Nino‐Vasquez et al .…”