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

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

doi:10.1007/s004380050930

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

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

Neuroscience

Producción científica: Article › revisión exhaustiva

28 Citas (Scopus)

Resumen

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.

Idioma original	English
Páginas (desde-hasta)	569-573
Número de páginas	5
Publicación	Molecular and General Genetics
Volumen	260
N.º	6
DOI	https://doi.org/10.1007/s004380050930
Estado	Published - 1999

Nota bibliográfica

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.

Financiación

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.

Financiadores
Bay Zoltán Foundation for Applied Research
Ministry of Culture and Education of Hungary

ASJC Scopus subject areas

Genetics

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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.",

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N1 - 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.

PY - 1999

Y1 - 1999

N2 - 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.

AB - 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.

KW - Abasic primer

KW - PCR cloning

KW - Sticky ends

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

Resumen

Nota bibliográfica

Financiación

ASJC Scopus subject areas

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