Optimization of direct whole blood PCR amplification with applications on a static thermostat chip.
In this paper, direct whole blood PCR amplifications on a static chip thermostat without sample purifications are demonstrated; in these amplifications, problems such as cross-interferences and contaminations could be avoided. The amplification conditions, such as the compositions of reagents and thermal programs, were investigated systematically by a GeneAmp PCR system with a native p53 gene segment (about 543 bp) of human genome and an exterior lambda DNA segment (about 500 bp) as targets. Direct amplifications of p53 and K-ras (about 157 bp) gene segments from 0.5 microL blood samples were successfully demonstrated by a static PCR chip with an indium tin oxide glass substrate. The chip thermostat has a typical size of 25 mm x 25 mm, and a polyethylene tube was used as the PCR vial on the glass surface of the chip. Fuzzy proportional integration-differentiation algorithms were adopted in temperature controls of the chip with an aid of a micro-Pt100 sensor. In the direct PCR with the thermostat chip, the whole process only involves automatic thermal programs. This work demonstrated that a chip PCR for field test without desktop facilities is possible either for a point of care test or for forensic analysis.
Qu
Bai-Yan
BY
Research Center of Analytical Science, Northeastern University, Wenhua Road 3-11, Shenyang 110004, China.
Wu
Zhi-Yong
ZY
Tian
Xiao-Xi
XX
Chen
Kun
K
Fang
Fang
F
D001769:Blood; D016158:Genes, p53 / Q000235:genetics; D046208:Microchip Analytical Procedures; D016133:Polymerase Chain Reaction / Q000379:methods*; D013696:Temperature; D018631:ras Proteins / Q000235:genetics
D018631:ras Proteins