Download Dr T-J’s Minilecture - Susquehanna University

Dr T-J’s Minilecture
Chapter 12
Restriction nuclease cutting followed by
ligation of sticky ends creates closed
circles from linear DNA fragments
Restriction nuclease cutting may generate
sticky (with overhangs)- or blunt-ends
DNA fragments may be amplified (cloned) by
joining with plasmid DNA and replication
of the recombinant DNA in bacteria
Foreign DNA and vector DNA both
must have matching sticky ends
Size limits of foreign DNA that can be
inserted into different cloning vectors
Other Vectors: BACs and
YACs
Different DNA fragments created by a restriction
nuclease may be joined in many different
arrangements since they all have the same sticky
ends
RNA templates may be copied into double
stranded DNA and then cloned
[complementary DNA (cDNA) cloning]
After being
copied into
DNA, the
RNA template
is usually
destroyed
(rather than
displaced)
before the
synthesis of
the second
DNA strand.
Useful features of a plasmid cloning
vector
Use of lacZ a-peptide coding sequence for
color-dependent selection of recombinant
clones
Use of a radioactive probe and
hybridization to immobilized DNA on a
filter for selection of desired clones
Contigs - Assembling full
sequences from smaller parts
Use of DNA microarrays (chips)
Fluorescently tagged
cDNA probes are
hybridized to DNA spots
in the microarray for
studying
differential expression of
thousands of genes at a
time in two mRNA
samples
Steps in the creation of a transgenic
mouse
Methodology for gene knockout or gene
replacement using a “targeting” vector
Site-specific mutagenesis of a cloned DNA
sequence using a synthetic mutagenic primer