Accelerated screening of cDNA libraries using the polymerase chain reaction and Southern blotting.
cDNA libraries are normally constructed in either phage or plasmid vectors and screened for sequences of interest using antibodies or, more commonly, nucleic acid probes. To clone a sequence of interest from a library generally involves at least three rounds of hybridization with 32P-labeled probes. This approach is highly labor intensive, and no information about the size of the hybridizing insert is obtained until the clones have been purified and the insert DNA analyzed by restriction enzyme digestion. We report on a rapid screening protocol for libraries constructed in bacteriophage lambda vectors involving polymerase chain reaction amplification of the insert from hybridizing phage plaques and on its analysis by agarose gel electrophoresis and Southern blotting. This can take place after only one round of conventional screening, and phage from a large number of positively hybridizing plaques can be analyzed by a "one-tube" reaction.
Fraser
D C
DC
AFRC Roslin Institute, Roslin, Midlothian, Scotland.
Craigmile
S
S
Russell
G C
GC
D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D015139:Blotting, Southern; D002417:Cattle; D018076:DNA, Complementary / Q000235:genetics*; D005822:Genetic Vectors; D015698:Genomic Library; D016133:Polymerase Chain Reaction
D018076:DNA, Complementary