Length and sequence variation in the apolipoprotein B intron 20 Alu repeat.
Length and sequence variation in the apolipoprotein B intron 20 Alu repeat.
复制标题
载脂蛋白 B 内含子 20 Alu 重复序列的长度和序列变异。
DOI：
10.1016/s0888-7543(05)80240-x
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发表时间：
1992
期刊：
影响因子：
4.4
通讯作者：
Boerwinkle,E
中科院分区：
文献类型：
--
作者：
Shriver,MD;Siest,G;Boerwinkle,E
我们建立了一种单链构象多态(SSCP)方法，用于检测位于载脂蛋白B(Apo B)基因内含子20的AluElement的序列和长度变异。通过聚合酶链式反应，我们同时扩增并同位素标记了载脂蛋白B内含子20Alu。用PstI限制性内切酶从其余的PCR产物中分离出Aluail，它由两个数目可变的串联重复序列(TTTX)y(X=A或G)和(T)z组成。因此，Aluail(in 20-rep)中的长度变异与Alubody(in 20-SEQ)中的序列变异是分开的，这使得SSCP模式更易于解释，信息也更丰富。在法国南希242名无关个体的样本中，我们在IN20-SEQ基因座上观察到11个SSCP等位基因，它们只是在序列上不同。在in20-rep基因座上，我们观察到7个在序列和长度上都不同的等位基因。对两个座位的等位基因进行亚克隆和测序。在对SSCP等位基因进行测序的过程中，在每个基因座上都发现了另外一个没有在SSCP凝胶中经历可检测到的迁移率变化的等位基因。用限制性内切酶对附加的20-SEQ等位基因进行分型。虽然in20-SEQ和in20-rep等位基因的数量很多，但大多数是不常见的；每个座位上最常见的三个等位基因占样本的94%以上。我们还对242名无血缘关系的法国人的子女进行了分型，从而验证了两个基因座的孟德尔分离，并构建了单倍型。在84个可能的单倍型中观察到23个，杂合度为0.813。不出所料，这两个基因座距离很近，处于显著的连锁不平衡状态。利用最大的简约性，我们能够毫不含糊地将12个IN20-SEQ等位基因中的11个置于系统发育网络中。我们的结论是，长度和序列多态是广泛和广泛分布的变异的来源，用于各种遗传应用。
We have developed a single-stranded conformation polymorphism (SSCP) protocol for typing both sequence and length variations in anAluelement located in intron 20 of the human apolipoprotein B (apo B) gene. Using the polymerase chain reaction (PCR), we simultaneously amplified and isotopically labeled the apo B intron 20Alu. TheAlutail, which is composed of two arrays of variable numbers of tandem repeats, (TTTX)y(X= A or G) and (T)z, was separated from the rest of the PCR product by restriction enzyme digestion withPstI. Length variation in theAlutail (IN20-REP) was thus separated from sequence variation in theAlubody (IN20-SEQ), rendering the SSCP patterns both easier to interpret and more informative. In a sample of 242 unrelated individuals from Nancy, France, we observed 11 SSCP alleles at the IN20-SEQ locus that differed only in sequence. At the IN20-REP locus, we observed 7 alleles that differed in both sequence and length. All alleles at both loci were subcloned and sequenced. One additional allele that did not undergo a detectable mobility shift in SSCP gels was uncovered at each locus during sequencing of the SSCP alleles. The additional IN20-SEQ allele was typed by restriction enzyme digestion. Although the number of IN20-SEQ and IN20-REP alleles was large, most were uncommon; the three most common alleles at each locus represented more than 94% of those sampled. We also typed the children of the 242 unrelated French individuals, enabling verification of the Mendelian segregation of the two loci and construction of haplotypes. Twenty-three out of a possible 84 haplotypes were observed with a heterozygosity of 0.813. As expected given their close proximity, these two loci are in significant linkage disequilibrium. Using maximum parsimony, we were able to unambiguously place 11 of the 12 IN20-SEQ alleles on a phylogenetic network. We conclude thatAlulength and sequence polymorphisms are a source of extensive and widely dispersed variation for a variety of genetic applications.
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