Detection and evaluation of non-recombinants in cDNA libraries by multiple cloning region PCR.
Bacteriophages that are routinely used in cDNA libraries do not require any biological selection for forming plaques. Thus parental non-recombinant phages are always found in variable proportions together with recombinant ones in all cDNA libraries. The presence of non-recombinants in significant proportions dilutes the abundance of rare cDNA species and makes library screening difficult. If the exact proportion of non-recombinants in a library were known, then one would screen proportionately more plaques to get a positive clone. In the absence of such information, screening is conventionally conducted on a number that is based on the titer of the library. We have devised a method using the flanking sequences from either side of the multiple cloning region (MCR) of all lambda phage vector derivatives as primers for PCR amplification. A non-recombinant phage produces a fragment equal to the size of the MCR, whereas a recombinant phage produces a fragment larger than the MCR, which is an MCR+ fragment. All cDNA libraries that we have studied show the presence of the MCR fragment (indicating non-recombinants) at variable proportions ranging between 6% and 36% of the total phages present. We also show that their presence negatively influences the retrieval of target cDNA sequences.
Bandyopadhyay
Dilip
D
Division of Laboratory Medicine, Tata Memorial Hospital, Parel, Mumbai, India. dilipsnb@vsnl.com
Vesuna
Farhad D
FD
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D018076:DNA, Complementary; D005069:Evaluation Studies as Topic; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D011995:Recombination, Genetic
D017931:DNA Primers; D018076:DNA, Complementary