Cloning strategies - Principles of Gene Manipulation - page 96

mRNA

5' AAA n 3'

Oligo (dT)

AAA n 3'
TTT n

5'

Tc R

Pst I

pBR322 (1) Reverse

transcriptase
(2) Alkali

Ap R

First strand synthesis

TTT n 5'

cDNA

3'

Pst I

Second strand synthesis

DNA polymerase
+ 4 dNTPs

5'
3' G

3' ACGTC CTGCA 3'

Terminal
transferase
+ dGTP

S1-nuclease

3'

3'

Terminal
transferase
+ dCTP

G
GG n GACGTC CTGCAGG n G

CC n C

CC n C

Annealing

cDNA

G CC n C GG n GACGTC

G

CTGCAGG n G CC n G

Select
Tc R , Ap S

Transformation host repairs
gaps, reconstructs Pst I sites

GACGTCC n C GG n GACGTC

CTGCAGG n G CC n CTGCAG

Fig. 6.5 An early cDNA cloning strategy, involving hairpin-primed second-strand DNA synthesis and homopolymer tailing to
insert the cDNA into the vector.

sequences) in the resulting library. This can be
addressed through the use of random oligonucleo-
tide primers, usually hexamers, for both first- and
second-strand cDNA synthesis. However, while this
eliminates 3′-end bias in library construction, the
resulting clones are much smaller, such that full-

length cDNAs must be assembled from several
shorter fragments. Secondly, as the size of a cDNA
increases, it becomes progressively more difficult to
isolate full-length clones. This is partly due to
deficiencies in the reverse-transcriptase enzymes
used for first-strand cDNA synthesis. The enzymes