The method of single-nucleotide variations detection using capillary electrophoresis and molecular beacons.
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.
Wang
Jinhui
J
Department of Clinical Laboratory, The Second Affiliated Hospital of Harbin Medical University, Harbin, China.
Wang
Wei
W
Liu
Yanhong
Y
Duo
Libo
L
Huang
Lijuan
L
Jiang
Xiaofeng
X
D001483:Base Sequence; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004586:Electrophoresis; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D017422:Sequence Analysis, DNA; D044366:Transition Temperature
D015342:DNA Probes