4-3.3. Commonly used vectors for cDNA cloning and expression

Vector	Features
Lambda gt10, Lambda gt11	DNA inserts of 7.6 kb and 7.2 kb, respectively, inserted at a unique EcoRI cloning site; recombinant Lambda gt10 selected on the basis of plaque morphology; Lambda gt11 has E. coli LacZ gene: LacZ and cDNA encoded protein is expressed as fusion protein.
Lambda ZAP series (phasmids)	Up to 10 kb DNA insert; therefore, most cDNAs can be cloned; polylinker has six cloning site; T3 and T7 RNA polymerase sites flank the polylinker so that riboprobes of both strands can be prepared; these features are contained in plasmid vector p Bluescript, which is inserted into the phage genome; the plasmid containing cDNA recovered simply by co-infecting the bacteria with a helper f1 phage that helps excise from the phage genome.

4-3.4. Problems in cDNA preparation

•  Large mRNA sequence results in inefficient synthesis of full- length cDNA. This cause problems during expression as it may not contain the entire coding sequence of the gene. This arises because of the poor processivity of RTase purified from avian myeloblastosis virus (AVM) or produced in E.coli from the gene of Moloney murine leukemia virus (MMLV).

•  Use of S1 nuclease, the enzyme used to trim the ds cDNA, may remove some important 5´ sequences.

4-3.5. Strategies to overcome the limitations in cDNA preparation

Strategies that can be employed to overcome the above limitations are listed as follows-

•  A specially designed E. coli vector can be used to avoid incomplete copying of the RNA.

•  The use of single strand specific nuclease can be avoided by adding a poly-C tail to the 3´-end of the single stranded cDNA produced by copying of the mRNA by the enzyme terminal deoxynucleotidyl transferase. Complementary oligonucleotide (Poly-G) is now used as a primer for the synthesis of complementary strand to yield ds-cDNA without a hairpin loop enhancing the full-length cDNA production.