Polymerase chain reaction of genes flanked by short noncontiguous sequence motifs.
Short DNA sequence motifs have been demonstrated to interact with DNA binding proteins and regulate flanking genes. The short nature and the lack of continuity of many of these DNA binding sites make it difficult to develop an approach to characterize genes that have the same flanking sequences. We tested various oligonucleotide combinations using an immunoglobulin variable region gene family as a model amplification system. One successful amplification strategy used an oligonucleotide containing two known noncontiguous short sequences connected by random insertion of all four bases to maintain the appropriate spacing. A second approach used an oligonucleotide having a single short homologous sequence with the addition of all four bases randomly placed at the 5' end to increase the extent of homology. Both strategies will permit the priming of members of a specific gene family, with the two short sequences bridged by all four bases randomly added being more efficient in the amplification process.
Borghesi-Nicoletti
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Department of Microbiology and Immunology, University of Maryland, Baltimore 21201.
Schulze
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DH
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D004247:DNA; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012045:Regulatory Sequences, Nucleic Acid
D004247:DNA