Roberts C H, Madrigal J A, Marsh S G E
Anthony Nolan Research Institute and Department of Haematology, Royal Free and UCL School of Medicine, London, UK.
Tissue Antigens. 2007 Apr;69 Suppl 1:88-91. doi: 10.1111/j.1399-0039.2006.762_2.x.
Killer-cell immunoglobulin-like receptor (KIR) genes are highly polymorphic and polymorphisms have been found in all KIR exons. Although less is known of the introns, these also appear to be polymorphic. To generate a comprehensive database of KIR genomic sequences, which will aid in the design of KIR typing reagents, we have established a method for cloning and sequencing of KIR genes from genomic DNA. We cloned and sequenced the entire KIR2DL4 gene from genomic DNA using long template touchdown PCR and high capacity cloning vectors. Overlapping secondary amplicons were modified to include a nucleotide analogue that reduced sequencing problems associated with secondary structure formation in the KIR sequence. Using a modified sequencing chemistry we were able to sequence approximately 11,000 bases confirming the previously published KIR2DL4*005 allele sequence.
杀伤细胞免疫球蛋白样受体（KIR）基因具有高度多态性，并且在所有KIR外显子中均发现了多态性。尽管对内含子的了解较少，但它们似乎也具有多态性。为了生成一个全面的KIR基因组序列数据库，以辅助KIR分型试剂的设计，我们建立了一种从基因组DNA中克隆和测序KIR基因的方法。我们使用长模板降落PCR和高容量克隆载体从基因组DNA中克隆并测序了整个KIR2DL4基因。对重叠的二级扩增子进行了修饰，使其包含一种核苷酸类似物，该类似物减少了与KIR序列中二级结构形成相关的测序问题。使用改良的测序化学方法，我们能够对大约11,000个碱基进行测序，从而确认了先前发表的KIR2DL4*005等位基因序列。
