Gene Cloning

Gene Identification and DNA Libraries 105
(a)
(b)
(c)
(d)
(e)
5'
mRNA
AAAAA 3'
3' TTTTT 5'
Reverse transcriptase
5'
3'
RNAse H
DNA ligase
mRNA
cDNA
5' 3' 5' 3' 5'
3'
cDNA
DNA polymerase I
5'
3'
5'
3'
AAAAA 3'
TTTTT 5'
AAAAA 3'
TTTTT 5'
AAAAA 3'
TTTTT 5'
AAAAA 3'
TTTTT 5'
Figure 4.13 Synthesis of cDNA. a) After addition of a polyT oligonucleotide
primer, which will anneal to the polyA tail of the mRNA, reverse transcriptase
can synthesize complementary DNA using the mRNA as template b). RNAse H
is added which will partially remove the RNA strand c), short regions of RNA
remain annealed which can act as primers for DNA synthesis by DNA
polymerase. The strand displacing activity of DNA polymerase I will ensure that
the RNA primers are removed d). Finally DNA ligase is used to mend any nicks
in the new DNA strand e).
different mRNA molecules allowing reverse transcriptase to synthesize a
strand of DNA complementary to each of the mRNAs in the cell. You now
need to replace the mRNA template with DNA; this can be achieved using
the enzyme RNAse H which selectively breaks down RNA in an RNA–DNA
duplex. A second DNA strand is synthesized by DNA polymerase using
short pieces of RNA which remain as primers.
Q4.17. List, in order, the enzymes you would need to use to make cDNA
from an mRNA template. In each case say what the enzyme is used for.
4.19 Cloning the cDNA Product
You now have your cDNA. To make a cDNA library, this needs to be cloned
into a vector and introduced into E. coli. Bacteriophage λ vectors are commonly
used for the construction of cDNA libraries because of the high efficiencies
associated with these vectors. As you will be cloning relatively
small, gene-sized pieces of DNA you can use an insertion vector (Figure
4.6a), but if you are constructing an expression library you will need to use