[Study on the genotyping of single nucleotide polymorphisms for a large number of samples by three-dimensional polyacrylamide gel-based microarray method].
OBJECTIVE
To genotype single nucleotide polymorphisms (SNPs) in a large number of samples by applying three-dimensional polyacrylamide gel-based microarray.


METHODS
The method relies on copolymerization of acrylamide-modified PCR products with acrylamide monomers and acryl-modified slides to prepare gel-based microarray. Then array is hybridized with a pair of specific probes and the two universal dual-color fluorescent detectors labeled with Cy3 or Cy5 respectively (Tag1 and Tag2). Electrophoresis is used in post-hybridization to remove the nonspecifically bound targets and mismatches. Finally, genotyping is based on the images captured through two-color fluorescent scanning.


RESULTS
The 3-D gel-immobilization of nucleic acids has a high immobilization yield and good hybridization efficiency. As universal dual-color fluorescent detectors are used, it is not required that specific probes be labeled for all SNPs, therefore the expense for synthesis can be reduced considerably. Electrophoresis in post-hybridization can enhance the capability for discriminating a single nucleotide mismatch from the perfectly matched sequence and improve the signal-to-noise ratio significantly.


CONCLUSIONS
The gel-based microarray is a rapid, simple and high-throughput method for SNPs genotyping and may be very competitive in the efficiency, fidelity and cost for constructing DNA microarrays, which will hold significant promise for applications in human DNA diagnostics.
Cheng
Lu
L
State Key Laboratory of Bioelectronics, School of Biological Science & Medical Engineering, Southeast University, Nanjing, Jiangsu, 210096 PR China.
Xiao
Peng-feng
PF
Sun
Bei-li
BL
Ge
Qin-yu
QY
Lu
Zu-hong
ZH
D004252:DNA Mutational Analysis / Q000379:methods*; D015342:DNA Probes; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005456:Fluorescent Dyes; D005838:Genotype; D006801:Humans; D025202:Molecular Diagnostic Techniques; D009693:Nucleic Acid Hybridization / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide
D015342:DNA Probes; D005456:Fluorescent Dyes