For the sake of simplicity, Fig. 10.4 omitted one step of cDNA library construct

Question

For the sake of simplicity, Fig. 10.4 omitted one step of cDNA library construction. The figure implied that the last step of the process is the ligation of bluntended cDNAs into plasmid cloning vectors. Although such ligation reactions can occur, in reality they are highly inefficient. Instead, scientists convert blunt-ended cDNA molecules into stick-ended molecules using adapters, and then they ligate the cDNAs into vectors with compatible sticky ends. Adapters are short, partly double-stranded DNA molecules made by hybridization of two single-stranded oligonucleotides made in a DNA synthesizer. Suppose that the following two oligonucleotides were synthesized and then mixed togehter at highg concentration and at a temperature that promotoes hybridization of complementary DNA sequences:

5' CCCCCG 3'

5' AATTCGGGGG 3'

a. draw the hybridized DNA molecules. These are the adapters.

b. suppose you added the adapters and ligase enzyme to blunt-ended cDNAs at a very high molar ratio of adapters to cDNAs, so that each cDNA molecule is ligated to one adapter at each of its ends. Draw a picture of resulting cDNA molecule.

c. the particular adapters discussed in this problem allow the cDNAs to be ligated efficiently into a vector treated with a commonly used restriction enzyme listed in Table 9.1. Name this restriction enzyme.

Explanation / Answer

a) 5' CCCCCG 3'

3' GGGGGCAATTC 5'

The above is the hybridized DNA molecule

b) 5' CTTAACGGGGG---------- cDNA ----------CCCCCG 3'

3' GCCCCC---------- cDNA ----------GGGGGCAATTC '5

the above is the resulting cDNA molecule that is ligated to the adapter

c) GAATTC is the restriction site that is recognised by the enzyme EcoRI.

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