Functional cloning vectors for use in directional cDNA cloning using cohesive ends produced with T4 DNA polymerase

Joseph L. Kuijper, Kristine Wiren, Laura D. Mathies, Charles L. Gray, Frederick S. Hagen

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

This paper describes the construction of 'Prime' cloning vectors, which include phage λ and plasmid vectors useful for functional cloning in oocytes, yeast, and mammalian cells, and their use in a 'Prime' cloning system. The system takes advantage of the very active and precise 3′ exonuclease activity of T4 DNA polymerase to produce single-stranded (ss) ends (cut-back) of vector and insert DNA. This results in the highly efficient directional cloning of cDNA and PCR-amplified DNA. The system obviates the need to digest insert DNA with a restriction endonuclease to unveil cloning sites, and thus eliminates the chance of internal digestion of the insert DNA. The cloning of PCR-amplified DNA, which is sometimes difficult, is made routine with this system. The 'Prime' sequence is included in vector cloning sites and cDNA and PCR primers. The 'Prime' sequence was chosen so that the ss sticky ends are nonpalindromic and will hybridize only to the appropriate partners. This makes cloning with the 'Prime' system very efficient, because neither the vector nor insert DNA is lost to unproductive self-hybridization.

Original languageEnglish (US)
Pages (from-to)147-155
Number of pages9
JournalGene
Volume112
Issue number2
DOIs
StatePublished - Mar 15 1992
Externally publishedYes

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Genetic Vectors
DNA-Directed DNA Polymerase
Organism Cloning
Complementary DNA
DNA
spleen exonuclease
Polymerase Chain Reaction
DNA Restriction Enzymes
Bacteriophages
Oocytes
Digestion
Plasmids
Yeasts

ASJC Scopus subject areas

  • Genetics

Cite this

Functional cloning vectors for use in directional cDNA cloning using cohesive ends produced with T4 DNA polymerase. / Kuijper, Joseph L.; Wiren, Kristine; Mathies, Laura D.; Gray, Charles L.; Hagen, Frederick S.

In: Gene, Vol. 112, No. 2, 15.03.1992, p. 147-155.

Research output: Contribution to journalArticle

Kuijper, Joseph L. ; Wiren, Kristine ; Mathies, Laura D. ; Gray, Charles L. ; Hagen, Frederick S. / Functional cloning vectors for use in directional cDNA cloning using cohesive ends produced with T4 DNA polymerase. In: Gene. 1992 ; Vol. 112, No. 2. pp. 147-155.
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