[通过聚合酶链反应检测常见α地中海贫血-2决定簇]

Zhao Y, Zhong M, Liu Z, Xu X

Department of Cell Biology and Medical Genetics, the First Military Medical University, Guangzhou, Guangdong 510515 P. R. China.

Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2001 Jun;18(3):216-8.

OBJECTIVE

Most frequent molecular lesions of alpha-thalassemia-2 determinants are deletions of one alpha-globin gene. Satisfactory PCR methodologies for detecting the deletions are required for molecular diagnosis and genetic screening since there was no internal control in most published PCR-based strategies. The purpose of this study is to develop a reliable PCR protocol specific for the two common alpha-thalassemia-2 determinants with internal control.

METHODS

The multiple repeat elements and the high GC-content of the alpha-globin locus impose severe limitations on designing suitable primers and optimizing stable conditions for PCR. In this study, two multiplex PCR systems were successfully set up. One was designed to detect the rightward deletion (-alpha(3.7)/) with two pairs of primers including one newly optimized pair for amplification of the internal standard to indicate the success of failure of PCR amplification. The other, to the leftward deletion(-alpha(4.2)/) with three primers, which were designed according to the newly sequenced data of the -alpha(4.2) and HbQ-alpha(4.2) deletions in this lab(Genbank Accession No. AF221717). In the PCR system, one is used as a common upstream primer and the other two are used as specific downstream primers for typing the normal allele and the deletion one, respectively.

RESULTS

Easily interpretable, unambiguous amplifications were observed by using the multiplex PCR systems for the detection of the two common alpha-thalassemia-2 determinants. The three or four primers were run in the same tube under the same condition and both of these two systems could be used at the same thermal cycle parameters. For typing the rightward deletion, a mutant-specific amplification of 1.7 kb and a 1140 bp amplified band as a normal and system control were produced. For typing the leftward deletion, two PCR-amplified bands, a 956 bp fragment specific for a -alpha(4.2) gene and a 1140 bp one for a normal allele were found.

CONCLUSION

Two sets of PCR systems with internal controls for detecting the most common two alpha-thalassemia-2 determinants have been established and may be suitable for molecular diagnosis and population screening.

目的

α地中海贫血-2决定簇最常见的分子病变是一个α珠蛋白基因的缺失。由于大多数已发表的基于聚合酶链反应（PCR）的策略中没有内部对照，因此分子诊断和基因筛查需要用于检测这些缺失的令人满意的PCR方法。本研究的目的是开发一种针对两种常见α地中海贫血-2决定簇且带有内部对照的可靠PCR方案。

方法

α珠蛋白基因座的多个重复元件和高GC含量对设计合适的引物以及优化PCR的稳定条件造成了严重限制。在本研究中，成功建立了两个多重PCR系统。一个用于检测向右缺失（-α(3.7)/），有两对引物，其中一对是新优化的用于扩增内标的引物，以指示PCR扩增的成败。另一个用于检测向左缺失（-α(4.2)/），有三条引物，这些引物是根据本实验室新测序的-α(4.2)和HbQ-α(4.2)缺失数据（Genbank登录号AF221717）设计的。在PCR系统中，一条用作共同的上游引物，另外两条分别用作鉴定正常等位基因和缺失等位基因的特异性下游引物。

结果

使用多重PCR系统检测两种常见α地中海贫血-2决定簇时观察到易于解释、明确的扩增。三条或四条引物在同一管中在相同条件下运行，并且这两个系统都可以在相同的热循环参数下使用。对于鉴定向右缺失，产生了1.7 kb的突变体特异性扩增条带以及作为正常对照和系统对照的1140 bp扩增条带。对于鉴定向左缺失，发现了两条PCR扩增条带，一条956 bp的片段特异性针对-α(4.2)基因，另一条1140 bp的片段针对正常等位基因。