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many of the most commonly used restriction enzymes. Linkers are blunt-end ligated

to the cDNA but since they are added much in excess of the cDNA the ligation process

is reasonably successful. Subsequently the linkers are digested with the appropriate

restriction enzyme which provides the sticky ends for efficient ligation to a vector

digested with the same enzyme. This process may be made easier by the addition of

adaptors rather than linkers which are identical except that the sticky ends are

preformed and so there is no need for restriction digestion following ligation (Fig. 6.7).

6.2.7 Enrichment methods for RNA

Frequently an attempt is made to isolate the mRNA transcribed from a desired gene

within a particular cell or tissue that produces the protein in high amounts. Thus if the

Blunt-ended DNA

5  pGGGATCCC

3  CCCTAGGGp

linker Blunt-ended ligation,using DNA ligase

Plasmid

5 

3 

5 

3 

pGGGATCCC

CCCTAGGG

GGGATCCC

CCCTAGGGp

pGATCCC

GG

GG

CCCTAGp

CTAG

p^5 pGATC

(^3) 
Ligation of cohesive
ends, using DNA ligase
Cut with
BamHI
Cut with
BamHI
G
A
T
C C
C
GG
G G
CCC
T
A
G
GA
T
C
C
A
TG Plasmid
containing
DNA insert
Fig. 6.7Use of linkers. In this example, blunt-ended DNA is inserted into a specific restriction site on
a plasmid, after ligation to a linker containing the same restriction site.
203 6.2 Constructing gene libraries