Highly multiplex and sensitive SNP genotyping method using a three‐color fluorescence‐labeled ligase detection reaction coupled with conformation‐sensitive CE

Abstract

For the development of clinically useful genotyping methods for SNPs, accuracy, simplicity, sensitivity, and cost-effectiveness are the most important criteria. Among the methods currently being developed for SNP genotyping technology, the ligation-dependent method is considered the simplest for clinical diagnosis. However, sensitivity is not guaranteed by the ligation reaction alone, and analysis of multiple targets is limited by the detection method. Although CE is an attractive alternative to error-prone hybridization-based detection, the multiplex assay process is complicated because of the size-based DNA separation principle. In this study, we employed the ligase detection reaction coupled with high-resolution CE-SSCP to develop an accurate, sensitive, and simple multiplex genotyping method. Ligase detection reaction could amplify ligated products through recurrence of denaturation and ligation reaction, and SSCP could separate these products according to each different structure conformation without size variation. Thus, simple and sensitive SNP analysis can be performed using this method involving the use of similar-sized probes, without complex probe design steps. We found that this method could not only accurately discriminate base mismatches but also quantitatively detect 37 SNPs of the tp53 gene, which are used as targets in multiplex analysis, using three-color fluorescence-labeled probes.

Recent Progress in Single-Nucleotide Polymorphism Biosensors

“…It has been reported that SNPs are frequently difficult to distinguish because of differences in only one base in the DNA sequence, corresponding to different alleles [ 16 ]. The specificity, sensitivity, and cost-effectiveness of method designs are decisive measures for improving SNP detection [ 17 , 18 ]. Moreover, it is crucial to exploit efficient assessment methods to detect SNPs in complicated genomes.…”

Simultaneous multiple single nucleotide polymorphism detection based on click chemistry combined with DNA-encoded probes

“…Mass spectrometry based methods 47 , 48 are limited by expensive instruments and the difficulty of the use of large-scale equipment for clinical testing. Capillary electrophoresis (CE) 49 – 51 has been used for the multiplex detection of nucleic acids. The capability of CE for multiplex detection is related to its highly effective separation ability, so it can easily achieve real high-throughput detection.…”

Ultrasensitive Multiplex Detection of Single Nucleotide Polymorphisms Based on Short-Chain Hybridization Combined with Online Preconcentration of Capillary Electrophoresis

“…Multiplex ligation-dependent probe amplification (MLPA) is a reliable and efficient technique for the detection of multiple gene mutations. , Specific probes with unique lengths were designed for different targets so that the PCR products of the ligated probes could be separated by CE. Another CE-based technique for multiple SNPs detection is primary PCR coupled with ligase detection reaction (LDR). , These CE-based assays have LODs of about 1–5% for multiple mutant alleles and are inadequate for early diagnosis testing.…”