摘要
目的研究KIR 2DL4基因测序分型中序列及分型结果"异常"的原因。方法 2016年5月对KIR 2DL4基因测序分型时检出的2例杂合碱基位置峰高不平衡、判定为模棱两可结果或无完全匹配分型结果的标本,采集新鲜外周血样,提取mRNA,反转录成c DNA后,进行c DNA分子克隆和单体型测序;同时采用荧光定量法检测基因组DNA中2DL4基因的拷贝数。结果分子克隆和单体型测序表明,1例标本携带正常的KIR 2DL4*00102,*00501及*011等位基因;另一标本中检出了KIR 2DL4*00102,*00501及*00602等位基因。2例标本中均检出了3种不同的KIR 2DL4等位基因,无新等位基因检出。荧光定量PCR实验证实这2例标本KIR 2DL4拷贝数均为3个。结论人类KIR单体型上KIR 2DL4基因的拷贝数存在多样性,当出现杂合碱基位置峰高不平衡、无与之完全匹配分型结果,并非由新等位基因所致。
Objective To explore the reason for unusual sequences and genotyping results in sequencing-based typing（ SBT） for KIR 2 DL4 gene. Methods Two samples with in-balance peak height at heterozygous nucleotide positions were observed during KIR 2 DL4 SBT,in which one sample was assigned with ambiguous SBT result and the other one was observed with no conclusive SBT results using the Assign 4. 7 software. We isolated total RNA from fresh peripheral blood and complementary DNA（ c DNA） transcripts was synthesized by RT-PCR,and then subjected to cloning and haplotype sequencing. The copy number of KR2 DL4 was determined by quantitative PCR reactions. Results The results of molecular cloning and haplotype sequencing demonstrated that one sample with three normal alleles KIR 2 DL4＊00102,＊00501,＊011; and the other sample with three normal KIR 2 DL4＊00102,＊00501 and＊00602 alleles were determined. No novel KIR 2 DL4 allele was identified. Both the two samples carry a copy number of 3 KIR 2 DL4 genes determined by quantitative PCR assay.Conclusion KIR 2 DL4 gene can carry extra diversity of copy number variation in human KIR haplotype,samples with inbalance peak height at heterozygous nucleotide positions and no conclusive result in KIR 2 DL4 SBT test can＇t be simply explained by putative novel alleles.
出处
《中国输血杂志》
北大核心
2017年第10期1106-1109,共4页
Chinese Journal of Blood Transfusion
基金
国家自然科学基金(81373158)
关键词
KIR
2DL4基因
测序分型
基因拷贝
杂合碱基
峰高
KIR 2D/A gene
sequencing-based typing
copy number
heterozygous nucleotide
peak height
