Functional cloning vectors for use in directional cDNA cloning using cohesive ends produced with T4 DNA polymerase.
This paper describes the construction of 'Prime' cloning vectors, which include phage lambda 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.
Kuijper
J L
JL
ZymoGenetics, Inc., Seattle, WA 98105.
Wiren
K M
KM
Mathies
L D
LD
Gray
C L
CL
Hagen
F S
FS
D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular / Q000379:methods*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D013604:T-Phages / Q000201:enzymology*
D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D004259:DNA-Directed DNA Polymerase