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Cloning and sequencing alleles of the KIR2DL4 gene from genomic DNA samples

Authors


Steven G. E. Marsh
Anthony Nolan Research Institute and
Department of Haematology
Royal Free and UCL School of Medicine
London
UK
Tel: 44 (20) 7284 8321
Fax: 44 (20) 7284 8331
e-mail: marsh@ebi.ac.uk

Abstract

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.

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