Download Molecular Cloning

Application in Molecular Cloning
David Shiuan
Department of Life Science,
Institute of Biotechnology and
Interdisciplinary Program of Bioinformatics
National Dong Hwa University
Molecular Cloning
In order to have enough DNA to work with for a single gene or
sequence, you must have a way to “clone”, or reproduce many
exact copies of that gene.
This is called “molecular cloning”
Gene of
interest
Plasmid Cloning Vector
1. Small
2. Stable in the chosen
host – usually E. coli
3. High copy number
4. Easy to purifiy
5. Can accommodate
foriegn DNA
6. Single “cloning” sites
7. Selectable marker –
antibiotic resistance
8. Easily introduced into
host (transformation or
transduction
Gene fusion systems
– monitor the activity
of a gene by fusing it
to another
Current favorites are the
autofluorescent proteins
HeLa cells expressing
gfp and rfp
Clontech website
YAC(Yeast
artificial
chromsome)
self-replicating
vector that can be
maintained in yeast
Can accommodate
large insert
Reeves et al., 1992, Methods Enzymol. 216:584-603
BAC (bacterial artificial
chromsomes)
Derived from the F plasmid of E. coli
low copy number (1-2 copies per
cell)
Shizuya et al, 1992, PNAS 89:9794-8797
Informatics for Molecular Cloning
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Restriction Enzyme Site Analysis
PCR Cloning –primer design
Codon Usage Analysis
Plasmid Construct – plasmid
drawing
PCR primer selection
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Primer Length
Melting Temperature (Tm)
Specificity
Complementary Primer Sequences
G/C content and Polypyrimidine (T, C) or
polypurine (A, G) stretches
3’-end Sequence
Primer 3
Codon Usage –
differ from organisms