HLA-DQA1 genotyping by using oligonucleotide microarrays / 中国实验血液学杂志

In order to fabricate the HLA-DQA1 genotyping chip and develop an integrated, parallel technical platform to type HLA system, a pair of primers and a set of probes were designed according to the sequences of HLA-DQA1 exon 2, where the polymorphism is concentrated. The oligonucleotide chip was made with the methods developed in our laboratory. The target DNA was asymmetrically amplified with the labeled sense primer. The signals were scanned and analyzed after the hybridization between microarray and PCR product. The allele types of the samples were identified. The result was verified by the standard DNA and DNA sequencing. The results showed that the genotyping was successfully carried out in 50 standard DNA samples and 50 clinical samples. Among them, results of the 50 standard DNA samples matched their templates. In the other 50 samples, results of the randomly selected 10 matched their sequencing results except that two of them got the incompletely result. In reproducible tests, the signal reappear rate was 95%. It is concluded that HLA-DQA1 genotyping by using our array system is simple and convenient with satisfied accuracy and reproducibility.

Quantitation and detection of deletion in tumor mitochondrial DNA by microarray technique / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To develop a method to rapidly quantitate and detect deletion of mitochondrial DNA (mtDNA) by microarray technique as a tool to study its relationship to tumorigenesis.</p><p><b>METHODS</b>A modified PCR was used to amplify full length mtDNA sequence in two samples of normal human blood leukocytes and five samples of gastric cancerous tissues, which were simultaneously labeled with fluorescin. The amplified products were verified by polyacrylamide gel electrophoresis (PAGE) and silver staining. Then, 17 pairs of overlapping primers of mtDNA were designed and their PCR products were used as mitochondrial probes. They were spotted onto amino-slides as microarray and hybridized. Hybridization image was scanned with GeneTAC laser, mtDNA copy number was counted by ScanAnalyzer software.</p><p><b>RESULTS</b>PAGE analysis showed that the designed probes were quite reasonable and strongly specific. The modified PCR method was efficient to amplify the whole mitochondrial genome with high-yield specific bands. The hybridizing spots were distinct, and background was clear. The signals of negative probes were close to those of background, and there was no significant difference between them (P > 0.05). The results were identical to those in the designed experiment. There were no significant differences between the results when the same sample of blood leucocytes or cancer tissues repeatedly examined with the same positive probes (P > 0.05), while there were significant differences when different types of samples were examined (P < 0.01). The hybridizing signals were stable and most of the data distributed in the range of mean +/- 2xSD.</p><p><b>CONCLUSION</b>The method here reported can rapidly, correctly and massively determine whether there exist special deletion and/or quantitative changes of mtDNA in patients with tumors. It will be helpful for the study of the relationship between mtDNA alteration and tumor development.</p>