New prediction model for probe specificity in an allele-specific extension reaction for haplotype-specific extraction (HSE) of Y chromosome mixtures.
Allele-specific extension reactions (ASERs) use 3' terminus-specific primers for the selective extension of completely annealed matches by polymerase. The ability of the polymerase to extend non-specific 3' terminal mismatches leads to a failure of the reaction, a process that is only partly understood and predictable, and often requires time-consuming assay design. In our studies we investigated haplotype-specific extraction (HSE) for the separation of male DNA mixtures. HSE is an ASER and provides the ability to distinguish between diploid chromosomes from one or more individuals. Here, we show that the success of HSE and allele-specific extension depend strongly on the concentration difference between complete match and 3' terminal mismatch. Using the oligonucleotide-modeling platform Visual Omp, we demonstrated the dependency of the discrimination power of the polymerase on match- and mismatch-target hybridization between different probe lengths. Therefore, the probe specificity in HSE could be predicted by performing a relative comparison of different probe designs with their simulated differences between the duplex concentration of target-probe match and mismatches. We tested this new model for probe design in more than 300 HSE reactions with 137 different probes and obtained an accordance of 88%.
Department of Forensic Genetics, Institute of Legal Medicine and Forensic Sciences, Charité- Campus Virchow-Klinikum, Berlin, Germany. jessica.rothe@charite.de
Jessica
J
Rothe
Norman E
NE
Watkins
Marion
M
Nagy
D000483:Alleles; D041322:Chromosomes, Human, Y; D003198:Computer Simulation; D004247:DNA / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D006239:Haplotypes; D006801:Humans; D008297:Male; D018895:Microsatellite Repeats / Q000235:genetics; D008957:Models, Genetic; D008962:Models, Theoretical; D009693:Nucleic Acid Hybridization / Q000235:genetics; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D004247:DNA; D004259:DNA-Directed DNA Polymerase