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目的检测汉族人群caspase10基因(CASP10)编码区外显子及剪接区域的多态性位点,研究CASP10基因与多基因复杂疾病的关联性。方法采用聚合酶链反应(polymerase chain reaction,PCR)、变性高效液相色谱技术(denaturing high-performanceliquid chromatography,DHPLC)、直接测序及克隆测序技术检测CASP10基因第9外显子及其部分侧翼序列。结果CASP10基因第9外显子在本人群70例血样本中未检测到存在于其他人群中已知的单核苷酸多态(single nucleotide polymorphism,SNP),但初步发现在第8内含子中靠近第9外显子处存在连续重复的单核苷酸T,且T的数目在不同的个体中存在差异。测序分析显示该处为一单核苷酸重复序列,提示该位点为一单核苷酸重复微卫星位点。经比较现存的基因组数据库,先前未有类似报道。进一步采用高保真酶扩增及测序都证实同样结果。用DHPLC法检测了70例浙江汉族人群血样本,结果均呈杂合状态,暂命名为IVS8-13(T)n。结论我们的结果提示汉族人群中该位点为一杂合度较高的单核苷酸重复的微卫星位点。这一结果与NCBI的dbSNP数据库中公布的该位点为一缺失型的SNP不同,提示这一位点的遗传差异可能与种族相关。汉族人群CASP10基因中存在的这个微卫星的意义还有待于深入研究。
Objective To detect polymorphisms of exon and splicing region of caspase10 (CASP10) coding region in Chinese Han population and to study the association between CASP10 gene and multiple genes complicated diseases. Methods The exon 9 and some flanking regions of CASP10 gene were detected by polymerase chain reaction (PCR), denaturing high-performance liquid chromatography (DHPLC), direct sequencing and cloning and sequencing. Results No single nucleotide polymorphism (SNP) was detected in exon 9 of CASP10 gene in 70 blood samples of our group. However, it was found that in the 8th intron There is a continuous repeat of single nucleotide T near the 9th exon, and the number of T is different in different individuals. Sequencing analysis showed that this site was a single nucleotide repeat sequence, suggesting that the site is a single nucleotide repeat microsatellite locus. Previously not similarly reported by comparison with existing genomic databases. The same result was confirmed by further amplification and sequencing of the high-fidelity enzyme. The DHPLC method was used to detect blood samples of 70 Han Chinese in Zhejiang Province. The results were heterozygous and temporarily named as IVS8-13 (T) n. Conclusions Our results suggest that this locus in the Han population is a single microsatellite repeat microsatellite locus with high heterozygosity. This result is different from that reported in NCBI’s dbSNP database for a missing SNP, suggesting that the genetic differences at this locus may be race-related. The significance of this microsatellite present in the CASP10 gene of Han population remains to be further studied.