Fast Multiplexed Polymerase Chain Reaction for Conventional and Microfluidic Short Tandem Repeat Analysis

:  The time required for short tandem repeat (STR) amplification is determined by the temperature ramp rates of the thermal cycler, the components of the reaction mix, and the properties of the reaction vessel. Multiplex amplifications in microfluidic biochip‐based and conventional tube‐based therma...

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Bibliographic Details
Published in:Journal of forensic sciences 2009-11, Vol.54 (6), p.1287-1296
Main Authors: Giese, Heidi, Lam, Roger, Selden, Richard, Tan, Eugene
Format: Article
Language:English
Subjects:
Deoxyribonucleic acid
DNA
DNA Fingerprinting - methods
forensic science
Forensic sciences
Humans
Medical equipment
Microfluidic Analytical Techniques
microfluidics
Molecular biology
nontemplate nucleotide addition
polymerase chain reaction
Polymerase Chain Reaction - instrumentation
Polymerase Chain Reaction - methods
Reproducibility of Results
short tandem repeat
stutter
Tandem Repeat Sequences
Temperature
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Summary::  The time required for short tandem repeat (STR) amplification is determined by the temperature ramp rates of the thermal cycler, the components of the reaction mix, and the properties of the reaction vessel. Multiplex amplifications in microfluidic biochip‐based and conventional tube‐based thermal cyclers have been demonstrated in 17.3 and 19 min, respectively. Optimized 28‐cycle amplification protocols generated alleles with signal strengths above calling thresholds, heterozygous peak height ratios of greater than 0.65, and incomplete nontemplate nucleotide addition and stutter of less than 15%. Full CODIS‐compatible profiles were generated using the Profiler Plus ID, COfiler and Identifiler primer sets. PCR performance over a wide range of DNA template levels from 0.006 to 4 ng was characterized by separation and detection on a microfluidic electrophoresis system, Genebench‐FX™. The fast multiplex PCR approach has the potential to reduce process time and cost for STR analysis and enables development of a fully integrated microfluidic forensic DNA analysis system.
ISSN:0022-1198
1556-4029
DOI:10.1111/j.1556-4029.2009.01200.x