Isolation of the 5'-flanking region of genes by thermal asymmetric interlaced polymerase chain reaction.
BACKGROUND
Isolation of the 5'-flanking region of gene segments adjacent to known sequences is a tedious task. Thermal asymmetric interlaced (TAIL)-PCR developed in 1995 uses nested sequence-specific primers together with a shorter arbitrary degenerate primer so that the relative amplification efficiencies of specific and nonspecific products can be thermally controlled. In the present study, we modified the TAIL-PCR method to isolate the 5'-flanking region of two human genes, human natriuretic peptide receptor type A (NPRA) and type B (NPRB) genes.


METHODS
We improved upon the original TAIL-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 PCR was used.


RESULTS
We succeeded in isolating the 5'-flanking region of the NPRA and NPRB genes directly from human genomic DNA.


CONCLUSIONS
Our TAIL-PCR method is applicable not only to be human genome but also to the genome of other life forms.
Nakayama
T
T
2nd Department of Internal Medicine, Nihon University School of Medicine, Ooyaguchi-kamimachi 30-1, Itabashi-ku, Tokyo 173-8610, Japan. tnakayam@med.nihon-u.ac.jp
Soma
M
M
Rahmutula
D
D
Ozawa
Y
Y
Kanmatsuse
K
K
D006162:Guanylate Cyclase / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D017461:Receptors, Atrial Natriuretic Factor / Q000235:genetics*; D019914:Taq Polymerase / Q000378:metabolism; D013696:Temperature; D000099242:Atrial Natriuretic Factor Receptor B
D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor; D019914:Taq Polymerase; D000099242:Atrial Natriuretic Factor Receptor B; C091715:atrial natriuretic factor receptor A