Facilitation of KIR genotyping by a PCR-SSP method that amplifies short DNA fragments
Facilitation of KIR genotyping by a PCR-SSP method that amplifies short DNA fragments
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DOI：
10.1111/j.1399-0039.2007.00923.x
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发表时间：
2007-11-01
期刊：
影响因子：
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通讯作者：
Estefania, E.
中科院分区：
文献类型：
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作者：
Vilches, C.;Castano, J.;Estefania, E.
1997年，通过序列特异性引物聚合酶链反应（PCR-SSP）检测杀伤细胞免疫球蛋白样受体（KIR）基因，发现人类基因组在它们编码的KIR上存在很大差异。虽然只有少数KIR基因在所有人类中是保守的，但大多数个体缺乏几个这样的基因，这些基因往往与不同的单倍型组合相关联。PCR-SSP技术，更新后用于检测最近发现的KIR基因和等位基因，仍然广泛用于分析人类种群的多样性，并研究KIR基因变异对人类健康的影响。已发表的几种用于KIR基因分型的PCR-SSP方法虽然简单且可靠，但缺点是依赖于0.5-2.0 kbp的DNA片段的扩增，而这种方法在低质量DNA中往往失败。有价值的dna收集往往包括这种质量差的样本，这导致数据和资源的损失。更糟糕的是，未被发现的假阴性或假阳性反应可能导致错误的基因频率和奇怪的基因组合。为了解决这些问题，我们重新设计了之前发表的KIR基因分型方法，使其产生短扩增子（大多数基因小于200 bp）。这种修饰使扩增失败最小化，从而赋予KIR基因分型更大的一致性和可靠性。此外，新的PCR-SSP方法检测到最近描述的几个KIR基因的等位基因，并允许在不增加反应数量的情况下区分KIR2DS4和KIR3DP1的主要结构变体。
Detection of killer-cell immunoglobulin-like receptors (KIR) genes by polymerase chain reaction with sequence-specific primers (PCR-SSP) led in 1997 to the discovery that human genomes diverge largely in the KIR they encode. While only a few KIR genes are conserved in all humans, most individuals lack several those genes, which tend to associate in diverse haplotypic combinations. The PCR-SSP technique, updated to detect the more recently identified KIR genes and alleles, is still used widely to analyze the diversity of human populations, and to study the influence of KIR-gene variability on human health. Several published PCR-SSP methods for KIR genotyping, although simple and robust, have the drawback of relying on the amplification of DNA fragments spanning 0.5-2.0 kbp, which tends to fail in low-quality DNAs. Valuable collections of DNAs often include such poor quality samples, which lead to loss of data and resources. Even worse, undetected falsely negative or positive reactions may result in erroneous gene frequencies and in odd gene combinations. To address those problems, we have redesigned our previously published KIR genotyping method so that it produces short amplicons (less than 200 bp for most genes). This modification minimizes amplification failures, thus conferring greater consistency and reliability to KIR genotyping. In addition, the new PCR-SSP method detects recently described alleles of several KIR genes, and allows for discrimination between the major structural variants of KIR2DS4 and KIR3DP1 without increasing the number of reactions.