Isolation of the 5'-flanking region of genes by thermal asymmetric interlaced polymerase chain reaction.
T Nakayama, M Soma, D Rahmutula, K Kanmatsuse, Y Ozawa
Med Sci Monit 2001; 7(3): BR345-349
BACKGROUND: Isolation of the 5'-flanking region of gene segments adjacentto known sequences is a tedious task. Thermal asymmetric interlaced (TAIL)-PCR developed in 1995 usesnested sequence-specific primers together with a shorter arbitrary degenerate primer so that the relativeamplification efficiencies of specific and nonspecific products can be thermally controlled. In the presentstudy, we modified the TAIL-PCR method to isolate the 5'-flanking region of two human genes, human natriureticpeptide receptor type A (NPRA) and type B (NPRB) genes.
MATERIAL AND METHODS: We improved upon the originalTAIL-PCR method in some aspects: three different series of eight AD primers were used simultaneously;a high denaturing temperature was used; and a Taq DNA polymerase developed for long and accurate PCRwas used.
RESULTS: We succeeded in isolating the 5'-flanking region of the NPRA and NPRB genes directlyfrom human genomic DNA.
CONCLUSION: Our TAIL-PCR method is applicable not only to be human genome butalso to the genome of other life forms.
Keywords: promoter, genomic cloning, hemispecific PCR, TAIL-PCR