Locus-specific vector/primer systems for rapid cloning of allelic variants.
We have developed a rapid cDNA cloning procedure which uses a single-stranded (ss) vector/primer in which the primer sequence is locus-specific. Vector/primers were constructed by substituting a specific oligodeoxynucleotide primer sequence in place of the polylinker in M13mp19. The ss vector/primer is linearized and used to prime cDNA synthesis. Recircularized DNA is then used directly to transform competent bacterial hosts. As no intermediate column purifications or extractions are necessary, the entire procedure is performed in a single tube, contributing to the overall simplicity of the protocol. The primary use for this kind of vector/primer system will be for cloning and sequencing multiple allelic variants of polymorphic loci which contain a conserved 3' sequence. The two vector/primers we report here are specific for HLA-DQ beta genes and for human Ig variable regions associated with IgM antibodies.
Hiraiwa
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Virginia Mason Research Center, Seattle, WA 98101.
Milner
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EC
D000483:Alleles; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics; D005803:Genes, Immunoglobulin; D014644:Genetic Variation; D005822:Genetic Vectors; D006683:HLA-DQ Antigens / Q000235:genetics; D059866:HLA-DQ beta-Chains; D009838:Oligodeoxyribonucleotides / Q000235:genetics
D006683:HLA-DQ Antigens; D059866:HLA-DQ beta-Chains; C057615:HLA-DQbeta antigen; D009838:Oligodeoxyribonucleotides; D004247:DNA