Genetics Objectives 14
1.
Restriction enzymes in cloning and Southern blotting: restriction enzymes cut
DNA at a specific site; those that cut at different places on each strand of the
double helix make “sticky” ends that are useful for incorporating cut strands of
DNA into a vector with the same restriction site. Thus, the DNA wanted can be
cloned in a vector, or can be run through a gel in a Southern blot to identify DNA
with the restriction site (or with restriction sites at specific lengths from each
other).
2.
Cloning vectors:
a.
Plasmid: up to 15 kb
 Advantages: can be used to make both genomic and cDNA libraries in
both bacteria and yeast
 Disadvantages: host does not take up plasmid often, limited in size
b.
Bacteriophage lambda: up to 20 kb
 Advantages: can be used to make both genomic and cDNA libraries,
host is infected efficiently with bacteriophage, and each bacteria can
produce hundreds of thousands of cloned DNA per lysed bacteria
 Disadvantages: limited in size
c.
Cosmid: up to 45 kb
d.
BAC: 100 to 300 kb
e.
YAC: 100 to 2000 kb
 Advantages: can incorporate large pieces of DNA
 Disadvantages: can’t make a cDNA library, can’t incorporate small
DNA fragments
3.
Genomic library: a genomic library is made by using DNA. The DNA is
digested and then incorporated into vectors to make a “library” of sequences in
the genome.
cDNA library: a cDNA library is made by taking mRNA and then reverse
transcribing it to make cDNA. This differs from a genomic library because it is a
library composed solely of the DNA of expressed genes (intronic sequences will
not be found in a cDNA library).