Mutation detection using ligase chain reaction in passivated silicon-glass microchips and microchip capillary electrophoresis.
The ligase chain reaction (LCR) following PCR is one of the most sensitive and specific methods for detecting mutations, especially single nucleotide polymorphisms (SNPs). Performing LCR in microchips remains a challenge because of the inhibitory effect of the internal surfaces of silicon-glass microchips. We have tested a dynamic polymer-based surface passivation method for LCR conducted in oxide-coated silicon-glass microchips. The combination of polyvinylpyrrolidone 40 (PVP-40) at 0.75% (w/v) with an excess of the ligase produced successful LCR in the silicon-glass microchips, with yields of ligated primers comparable to reactions performed in conventional reaction tubes. Ligated primers were detected and quantified simply and conveniently using microchip capillary electrophoresis.
University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
Xing Jian
XJ
Lou
Nicholas J
NJ
Panaro
Peter
P
Wilding
Paolo
P
Fortina
Larry J
LJ
Kricka
D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers / Q000032:analysis; D019075:Electrophoresis, Capillary; D005898:Glass; D006801:Humans; D022521:Ligase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D008214:Lymphocytes; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D011205:Povidone / Q000737:chemistry*; D012825:Silicon; D012822:Silicon Dioxide / Q000737:chemistry; D013499:Surface Properties
D017931:DNA Primers; D012822:Silicon Dioxide; D011205:Povidone; D012825:Silicon