Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Development and validation of the AmpFlSTR® Identifiler® Direct PCR Amplification Kit: a multiplex assay for the direct amplification of single-source samples.

Author(s): Wang DY, Chang CW, Lagacé RE, Oldroyd NJ, Hennessy LK

Publication: J Forensic Sci, 2011, Vol. 56, Page 835-45

PubMed ID: 21418220 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of storing blood and buccal cells on FTA cards and amplification parameters on the amplification of STR. Blood specimens for this study were purchased and the source of buccal cells was unspecified.

   Summary of Findings:

   Consistent amplification of DNA of FTA cards was obtained from specimens diluted by as much as 1:8 (100-200 cells). Peak heights, intralocus balance and signal were unaffected by storage duration. Complete concordance of genotypes was observed between FTA samples and purchased DNA. Increasing cycle numbers increased peak height, but when 28 or 29 cycles were used there were off-scale homozygote peaks for some specimens. The highest peak heights were obtained when denaturation was performed at 94 degrees C, rather than 92.5 or 95.5 degrees C. No loss of genotypes occurred when annealing temperatures were between 55 and 61 degrees C, but annealing at 63 degrees C led to decreased peak height. The use of a final extension of 5 min resulted in incomplete terminal nucleotide addition at 5 loci, but after 15 min all 15 loci were complete and no further benefit was found by increasing extension time.

   Biospecimens

   • Bodily Fluid - Blood
   • Cell - Buccal

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   DNA	Microsatellite analysis

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Time at room temperature	0 days 42 days 84 days 126 days 168 days 210 days
   Biospecimen Aliquots and Components	Aliquot size/volume	75-80 ul whole blood 1:4 dilution 1:8 dilution 1:16 dilution 1:32 dilution
   Microsatellite analysis Specific	Number of cycles	25 26 27 28 29
   Microsatellite analysis Specific	Nucleic acid amplification	Multiple denaturation temperatures Multiple annealing temperatures Multiple elongation times

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