| Autor: |
Shawn W, Forrest, Timothy D, Kupferschmid, Brant C, Hendrickson, Thaddeus, Judkins, Daniel J, Petersen, Thomas, Scholl |
| Rok vydání: |
2004 |
| Předmět: |
|
| Zdroj: |
Croatian medical journal. 45(4) |
| ISSN: |
0353-9504 |
| Popis: |
Two rare polymorphisms were identified at the primer annealing sites of the short tandem repeat (STR) loci D8S1179 and D13S317 for a commercial multiplex STR system commonly used for human identification. These polymorphisms resulted in weak amplification from the affected alleles. Therefore, alternative polymerase chain reaction (PCR) thermal cycling conditions were developed that promoted more even signal amplitudes from these alleles by employing reduced annealing temperatures.Genomic DNA was isolated from bloodstains on FTA paper or cotton cloth. Multiplex genotyping was performed using commercially available reagents. DNA sequences of the affected alleles were determined by using automated instruments. In separate experiments, 96 genetically diverse samples were sequenced to identify polymorphisms surrounding D8S1179 and D13S317.Sequencing the two STR loci, D8S1179 and D13S317, from heterozygous samples that displayed disproportionate signals between alleles revealed a single nucleotide polymorphism (SNP) in each locus that was coincident with the primer annealing sites. Adjusting the primer annealing temperature during the PCR effectively eliminated the amplification bias between alleles due to the mismatched base and improved data quality. No additional polymorphisms were detected at these loci from sequencing 96 genetically diverse samples.The technique reported here benefits forensic science practitioners who encounter severe imbalance of heterozygous peaks. This approach is very cost effective, can be used in high-throughput situations, and consumes minimal sample. Finally, understanding polymorphisms at STRs employed for human identification should assist the development of improved reagents to interrogate these loci. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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