Download - Dr T-J’s Minilecture
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 -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