The method of single-nucleotide variations detection using capillary electrophoresis and molecular beacons

Abstract

We demonstrate that single-nucleotide variations in a DNA sequence can be detected using capillary electrophoresis (CE) and molecular beacons (MBs). In this method, the region surrounding the site of a nucleotide variation was amplified in a polymerase chain reaction, then hybridize PCR products with each of MBs. The sequences of the PCR products are different at the site of 2,044 in exon of interleukin (IL)-13 which to be identified. Through denaturation, the PCR product became single strand and hybridized with the completely complementary MB. The MB-target duplexes were separated using CE and solution-based fluorescence techniques. The results show that in each reaction a fluorescent response was elicited from the molecular beacon which was perfectly complementary to the amplified DNA, but not from the other MB whose probe sequence mismatched the target sequence. The method of CE based on MBs is able to identify single-nucleotide variations in a DNA sequence and can discriminate the genotyping of the SNP between the homo- and heteroduplexes of DNA fragments.

Recent trends in molecular beacon design and applications

“…Capillary electrophoresis is a highly efficient separation technique, and has been used in the hybridization analysis of MBs with DNA targets [41,42], in which an effective separation of the MB-target hybrid from the unhybridized MB can be achieved. The separation of the MB-target hybrid avoids the interference from the signal from the unhybridized MB, eliminating false-positive results completely.…”

Separation of single‐base sequential isomers of single‐stranded DNA by capillary electrophoresis and its application in the discrimination of single‐base DNA mutations

“…The most recent SNP detection methods are based on the recognition of a target sequence by formation of a duplex between the target sequence and its specific probe, which has a sequence complementary to the target . CE‐based SNP detection and genotyping methods based on such duplex formation also have been reported . As described above, the separation of single‐base substituent can be achieved by our proposed method.…”

A novel microfluidic chip electrophoresis strategy for simultaneous, label-free, multi-protein detection based on a graphene energy transfer biosensor

“…[12][13][14] Very recently, the biological applications of GO, have been a very active research eld in the development of biosensors because of the ultrahigh quenching efficiency of GO. 2,4,[15][16][17][18][19] And then, in order to investigate the possibility of multi-detection based on chip-CE, in the same way as the literature, in this paper GO was anticipated to serve as an energy acceptor of the uorescein FRET system to construct a uorescent sensor for the sensitive detection of the biorecognition event between an aptamer and protein. Three kinds of proteins were chosen as target proteins considering that their aptamers had been utilized by others demonstrating their stronger binding affinity.…”