[The Rsa I and Sma I polymorphisms in the von Willebrand factor gene discrimination by hybridization with oligonucleotide arrays].
OBJECTIVE
To develop a new assay for the Rsa I and Sma I polymorphisms (single nucleotide polymorphism, SNP) in the von Willebrand factor gene detection that utilizes oligonucleotide arrays (DNA chips) on glass supports. Expoloring the association of the RsaI and SmaI polymorphisms in the von Willebrand factor gene with thrombosis in Chinese.


METHODS
Allele-specific oligonucleotides are covalently immobilized on glutaradehyde derivatized glass slides in arrays. Single strand PCR product of PCR-amplified genomic DNA is fluorescently labeled by assymetric PCR with fluorescently tagged dUTP and hybridized to the support-bound oligonucleotide array. The hybridization pattern is detected by fluorescence scenning. The effect of hybridization conditions were evaluated and optimized. The method was validated by the discrimination of blinded DNA samples identified by restriction enzymes. 50 patients with thrombosis were examined by this method.


RESULTS
The genotypes scored by oligonucleotide arrays assay were in 100% agreement with restriction enzymes results. The genotype distributions GG, GA, AA and CC, CT, TT of RsaI and SmaI polymorphisms of patients or controls were 4.0%, 12.0%, 84.0% and 24.0%, 44.0%, 32.0% or 1.4%, 11.8%, 86.8% and 8.8%, 57.4%, 33.8%. The allele frequencies G, A and C, T were 10.0%, 90.0% and 46.0%, 54.0% or 7.4%, 92.6% and 37.5%, 62.5%. No statistical difference in genotype distribution and allele frequencies was observed between patients with thrombosis and the control groups (all P > 0.05).


CONCLUSIONS
A rapid and accurate method for the RsaI and SmaI polymorphisms in the von Willebrand factor gene detection, or for the analysis of single nucleotide polymorphism (SNP) has been developed using oligonucleotide arrays on glass supports. No association was observed between the Rsa I and Sma I polymorphisms in the vWF gene and thrombotic disease.
Dai
K
K
Jiangsu Institute of Haemotology, First Affiliated Hospital of Suzhou University, Suzhou 215007, China.
Zhao
Y
Y
Wang
H
H
D000483:Alleles; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005787:Gene Frequency; D005838:Genotype; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D013927:Thrombosis / Q000235:genetics; D014841:von Willebrand Factor / Q000235:genetics*
D014841:von Willebrand Factor; D004247:DNA; C025651:CCCGGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; C040158:GTAC-specific type II deoxyribonucleases