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

Author

P, Fattorini, F, Cossutta, P, Giulianini, P, Edomi, and C, Previderè

Subjects
HLA Antigens, Humans, Polymerase Chain Reaction, Sensitivity and Specificity, Alleles, DNA Damage, DNA Primers
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.

Published
2000

2. Subtyping mtDNA haplogroup H by SNaPshot minisequencing and its application in forensic individual identification.

Author

P. Grignani, G. Peloso, A. Achilli, C. Turchi, A. Tagliabracci, M. Alù, G. Beduschi, U. Ricci, L. Giunti, C. Robino, S. Gino, and C. Previderè

Abstract

Sequence variation of the hypervariable segments (HVS) I/II of mitochondrial DNA (mtDNA) and the haplogroup affiliation were determined in a sample of 271 Italian subjects. This analysis showed that 42% of the individuals could be ascribed to H, the most frequent haplogroup in European Caucasian populations. This fraction was then screened for specific single nucleotide polymorphisms located in the coding region to identify H subclades H1–H15. We set up two multiplex polymerase chain reactions and specific SNaPshot assays to investigate the frequency distribution of these subgroups in our population sample and to examine their usefulness in discriminating among commonly shared HVS I/II sequences. This allowed the assignment of a large portion of the mtDNAs (∼70%) to specific subhaplogroups, with H1 and H5 being the most represented. About two-thirds of the individuals sharing common HVS I/II sequences were subdivided and ascribed to specific H subhaplogroups with a significant reduction of the frequencies of the most common mtDNA haplotypes. Haplogroup H subtyping could thus be extremely useful in forensic identification when many samples have to be analysed and compared, avoiding excessive time-consuming and labor-intensive sequencing analysis. [ABSTRACT FROM AUTHOR]

Published
2006

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