Directional cloning of native PCR products with preformed sticky ends (Autosticky PCR)

被引：27

作者：

Gál J. ^{[1
,2
]}

Schnell R. ^{[1
,2
]}

Szekeres S. ^{[1
,2
]}

Kálmán M. ^{[1
,2
]}

机构：

[1] Institute for Biotechnology, Bay Zoltan Found. for Appl. Res., P.O. Box 2337

[2] Institute of Genetics, Biol. Res. Center of the Hungarian, Academy of Sciences, Szeged

来源：

Molecular and General Genetics MGG | 1999年 / 260卷 / 6期

关键词：

Abasic primer; PCR cloning; Sticky ends;

D O I：

10.1007/s004380050930

中图分类号：

学科分类号：

摘要：

A novel method for the directional cloning of native PCR products was developed. Abasic sites in DNA templates make DNA polymerases stall at the site during synthesis of the complementary strand. Since the 5' ends of PCR product strands contain built-in amplification primers, abasic sites within the primers result in the formation of 5' single-stranded overhangs at the ends of the PCR product, enabling its direct ligation to a suitably cleaved cloning vector without any further modification. This 'autosticky PCR' (AS-PCR) overcomes the problems caused by end sensitivity of restriction enzymes, or internal restriction sites within the amplified sequences, and enables the generation of essentially any desired 5' overhang.

引用

页码：569 / 573

页数：4

共 40 条

[1]

Ailenberg, M., Silverman, M., Description of a one step staggered reannealing method for directional cloning of PCR-generated DNA using sticky-end ligation without employing restriction enzymes (1996) Biochem Mol Biol Int, 39, pp. 771-779

[2]

Aslanidis, C., De Jong, P.J., Ligation-independent cloning of PCR products (LIC-PCR) (1990) Nucleic Acids Res, 18, pp. 6069-6074

[3]

Borovkov, A.Y., Rivkin, M.I., Xcml-containing vector for direct cloning of PCR products (1997) Biotechniques, 22, pp. 812-814

[4]

Clark, J.M., Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases (1988) Nucleic Acids Res, 16, pp. 9677-9686

[5]

Costa, G.L., Weiner, M.P., Polishing with T4 or Pfu polymerase increases the efficiency of cloning of PCR fragments (1994) Nucleic Acids Res, 22, p. 2423

[6]

Hemsley, A., Arnheim, N., Toney, M.D., Cortopassi, G., Galas, D.J., A simple method for site-directed mutagenesis using the polymerase chain reaction (1989) Nucleic Acids Res, 17, pp. 6545-6551

[7]

Hevroni, D., Livneh, Z., Bypass and termination at apurinic sites during replication of single-stranded DNA in vitro: A model for apurinic site mutagenesis (1988) Proc Natl Acad Sci USA, 85, pp. 5046-5050

[8]

Holton, T.A., Graham, M.W., A simple and efficient method for direct cloning of PCR products using ddT-tailed vectors (1991) Nucleic Acids Res, 19, p. 1156

[9]

Horn, T., Urdea, M.S., A chemical 5′-phosphorylation of oligodeoxyribonucleotides that can be monitored by trityl cation release (1986) Tetrahedron Lett, 27, pp. 4705-4708

[10]

Hu, G., DNA polymerase-catalyzed addition of nontemplated extra nucleotides to the 3′ end of a DNA fragment (1993) DNA Cell Biol, 12, pp. 763-770

← 1 2 3 4 →