On-chip ligation of multiplexing probe-pairs for identifying point mutations out of dense SNP loci.
Detailed analyses of dense single nucleotide polymorphism (SNP) loci within rifampin-resistance determining region (RRDR) are very important for the early assessment of drug resistance of Mycobacterium tuberculosis. A strategy was developed here to specifically identify point mutations out of dense SNP loci by on-chip ligation of multiplexing probe-pairs (MPPs). A probe-pair combines a common probe with a discriminating probe which is covalently attached to a DNA chip. The common probe hybridizes to the discriminating probe via a unique "zip-code complement". The allele-specific part on the 3'-end of the discriminating probe becomes covalently ligated to the adjacent part on the 5'-end of the common probe if and only if a mutation is present. Thus upon zip-code recognition, the process of identifying a mutation of interest is entirely located into corresponding well on the chip. As a consequence, cross-reactions and biased competitive attachments to targets, both of which result from the presence of various multiplexing probes, are greatly minimized. Mutation detection was performed by direct visualization using enzyme-linked assay. The method was demonstrated with an initial set of 24 probe-pairs targeting 22 clinically meaningful mutations within an 81-bp RRDR. 130-bp fragments of the rpoB gene from 15 clinical isolates were identified and were in 100% agreement with results from independent sequencing.
Xu
Chao
C
State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Xiao Hong Shan, Wuchang, Wuhan 430071, PR China.
Zhou
Ya-Feng
YF
Deng
Jiao-Yu
JY
Deng
Xiang
X
Guo
Yong-Chao
YC
Cui
Zong-Qiang
ZQ
Zhang
Zhi-Ping
ZP
Wei
Hong-Ping
HP
Bi
Li-Jun
LJ
Zhang
Xian-En
XE
D001426:Bacterial Proteins / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000295:instrumentation* / Q000379:methods; D015342:DNA Probes / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases; D004867:Equipment Design; D019544:Equipment Failure Analysis; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation*; D017354:Point Mutation / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D001426:Bacterial Proteins; D015342:DNA Probes; D004269:DNA, Bacterial; C505805:rpoB protein, Mycobacterium tuberculosis; D012321:DNA-Directed RNA Polymerases