On-chip ligation of multiplexing probe-pairs for identifying point mutations out of dense SNP loci

Biosensors & Bioelectronics
Chao XuXian-En Zhang

Abstract

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 meanin...Continue Reading

References

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