Oligonucleotide ligation assay-based DNA chip for multiplex detection of single nucleotide polymorphism.
An oligonucleotide ligation assay-based DNA chip has been developed to detect single nucleotide polymorphism. Synthesized nonamers, complementary to the flanking sequences of the mutation sites in target DNA, were immobilized onto glass slides through disulfide bonds on their 5' terminus. Allele-specific pentamers annealed adjacent to the nonamers on the complementary target DNA, containing 5'-phosphate groups and biotin labeled 3'-ends, were mixed with the target DNA in tube. Ligation reactions between nonamers and pentamers were carried out on chips in the presence of T4 DNA ligase. Ligation products were directly visualized on chips through enzyme-linked assay. The effect of G:T mismatch at different positions of pentamers on the ligation were evaluated. The results showed that any mismatch between pentamer and the target DNA could lead to the decrease of ligation, which can be detected easily. The established approach was further used for multiplex detection of mutations in rpoB gene of rifampin-resistant Mycobacterium tuberculosis clinical isolates.
Deng
Jiao-Yu
JY
Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
Zhang
Xian-En
XE
Mang
Yuan
Y
Zhang
Zhi-Ping
ZP
Zhou
Ya-Feng
YF
Liu
Qiang
Q
Lu
Hong-Bing
HB
Fu
Zhi-Jun
ZJ
D004247:DNA / Q000032:analysis*; D011088:DNA Ligases; D015342:DNA Probes; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D009841:Oligonucleotides / Q000096:biosynthesis; D020641:Polymorphism, Single Nucleotide; D012293:Rifampin / Q000494:pharmacology
D015342:DNA Probes; D009841:Oligonucleotides; D004247:DNA; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit; D011088:DNA Ligases; D012293:Rifampin