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the poly(A) of mRNA. Alkaline hydrolysis is then used to remove the RNA strand,

leaving single-stranded DNA which can be used, like the mRNA, to direct the synthesis

of a complementary DNA strand. The second-strand synthesis requires an oligo(dG)

primer, base-paired with the poly(dC) tail, which is catalysed by the Klenow fragment

of DNA polymerase I. The final product is double-stranded DNA, one of the strands

being complementary to the mRNA. One further method of cDNA synthesis involves

the use of RNase H. Here the first-strand cDNA is carried out as above with reverse

transcriptase but the resulting mRNA–cDNA hybrid is retained. RNase H is then used

at low concentrations to nick the RNA strand. The resulting nicks expose 3^0 hydroxyl

groups which are used by DNA polymerase as a primer to replace the RNA with a

second strand of cDNA (Fig. 6.6).

6.2.6 Treatment of blunt cDNA ends

Ligation of blunt-ended DNA fragments is not as efficient as ligation of sticky ends,

therefore with cDNA molecules additional procedures are undertaken before ligation

with cloning vectors. One approach is to add small double-stranded molecules with

one internal site for a restriction endonuclease, termednucleic acid linkers, to the

cDNA. Numerous linkers are commercially available with internal restriction sites for

Messenger RNA

AAAAAA-3

Anneal primer (poly(dT))

Poly(dT) primer

Reverse transcriptase/buffer/dNTPs

5 

RNase H/DNA polymerase I

RNase H leaves gaps in mRNA strand

DNA polymerase I utilises primer–template complexes formed from RNase H

Double-stranded cDNA

AAAAAA-3

AAAAAA-3

AAAAAA-3

5 

5 

5 

5 

3 

5 

3 

5 

3 

3 

5 

Fig. 6.6Second-strand cDNA synthesis using the RNase H method.

202 Recombinant DNA and genetic analysis