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