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

J. Gál, R. Schnell, S. Szekeres, M. Kálmán

Research output: Contribution to journalArticlepeer-review

26 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)569-573
Number of pages5
JournalMolecular and General Genetics
Volume260
Issue number6
DOIs
StatePublished - 1999

Bibliographical note

Funding Information:
Acknowledgements We thank Dr. László Dorgai, Dr. István Ras-kó and Dr. Péter Putnoky for helpful discussions and Andrea Vörös for technical assistance. Our work was supported by grants from the Bay Zoltán Foundation for Applied Research and the Ministry of Culture and Education of Hungary. József Gál, Róbert Schnell and Szilvia Szekeres are Ph.D. students of the József Attila University, Szeged, Hungary. All experiments presented in this work were performed in compliance with the relevant laws of Hungary.

Keywords

  • Abasic primer
  • PCR cloning
  • Sticky ends

ASJC Scopus subject areas

  • Genetics

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