TY - JOUR
T1 - Internal validation of the GlobalFiler™ Express PCR Amplification Kit for the direct amplification of reference DNA samples on a high-throughput automated workflow
AU - Flores, Shahida
AU - Sun, Jie
AU - King, Jonathan
AU - Budowle, Bruce
PY - 2014/5
Y1 - 2014/5
N2 - The GlobalFiler™ Express PCR Amplification Kit uses 6-dye fluorescent chemistry to enable multiplexing of 21 autosomal STRs, 1 Y-STR, 1 Y-indel and the sex-determining marker amelogenin. The kit is specifically designed for processing reference DNA samples in a high throughput manner. Validation studies were conducted to assess the performance and define the limitations of this direct amplification kit for typing blood and buccal reference DNA samples on various punchable collection media. Studies included thermal cycling sensitivity, reproducibility, precision, sensitivity of detection, minimum detection threshold, system contamination, stochastic threshold and concordance. Results showed that optimal amplification and injection parameters for a 1.2 mm punch from blood and buccal samples were 27 and 28 cycles, respectively, combined with a 12 s injection on an ABI 3500xL Genetic Analyzer. Minimum detection thresholds were set at 100 and 120 RFUs for 27 and 28 cycles, respectively, and it was suggested that data from positive amplification controls provided a better threshold representation. Stochastic thresholds were set at 250 and 400 RFUs for 27 and 28 cycles, respectively, as stochastic effects increased with cycle number. The minimum amount of input DNA resulting in a full profile was 0.5 ng, however, the optimum range determined was 2.5-10 ng. Profile quality from the GlobalFiler™ Express Kit and the previously validated AmpFlSTR® Identifiler® Direct Kit was comparable. The validation data support that reliable DNA typing results from reference DNA samples can be obtained using the GlobalFiler™ Express PCR Amplification Kit.
AB - The GlobalFiler™ Express PCR Amplification Kit uses 6-dye fluorescent chemistry to enable multiplexing of 21 autosomal STRs, 1 Y-STR, 1 Y-indel and the sex-determining marker amelogenin. The kit is specifically designed for processing reference DNA samples in a high throughput manner. Validation studies were conducted to assess the performance and define the limitations of this direct amplification kit for typing blood and buccal reference DNA samples on various punchable collection media. Studies included thermal cycling sensitivity, reproducibility, precision, sensitivity of detection, minimum detection threshold, system contamination, stochastic threshold and concordance. Results showed that optimal amplification and injection parameters for a 1.2 mm punch from blood and buccal samples were 27 and 28 cycles, respectively, combined with a 12 s injection on an ABI 3500xL Genetic Analyzer. Minimum detection thresholds were set at 100 and 120 RFUs for 27 and 28 cycles, respectively, and it was suggested that data from positive amplification controls provided a better threshold representation. Stochastic thresholds were set at 250 and 400 RFUs for 27 and 28 cycles, respectively, as stochastic effects increased with cycle number. The minimum amount of input DNA resulting in a full profile was 0.5 ng, however, the optimum range determined was 2.5-10 ng. Profile quality from the GlobalFiler™ Express Kit and the previously validated AmpFlSTR® Identifiler® Direct Kit was comparable. The validation data support that reliable DNA typing results from reference DNA samples can be obtained using the GlobalFiler™ Express PCR Amplification Kit.
KW - Direct amplification
KW - Forensic STR loci
KW - GlobalFiler Express™
KW - Reference DNA samples
KW - Short tandem repeats
KW - Y-indel
UR - http://www.scopus.com/inward/record.url?scp=84894289476&partnerID=8YFLogxK
U2 - 10.1016/j.fsigen.2014.01.005
DO - 10.1016/j.fsigen.2014.01.005
M3 - Article
C2 - 24552885
AN - SCOPUS:84894289476
SN - 1872-4973
VL - 10
SP - 33
EP - 39
JO - Forensic Science International: Genetics
JF - Forensic Science International: Genetics
IS - 1
ER -