application in molecular cloning david shiuan department of life science, institute of biotechnology...
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Application in Molecular Cloning
David Shiuan
Department of Life Science, Institute of Biotechnology and
Interdisciplinary Program of Bioinformatics
National Dong Hwa University
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”
Molecular Cloning
Gene of interest
1. Small2. Stable in the chosen
host – usually E. coli3. High copy number4. Easy to purifiy5. Can accommodate
foriegn DNA6. Single “cloning” sites7. Selectable marker –
antibiotic resistance8. Easily introduced into
host (transformation or transduction
Plasmid Cloning Vector
Gene fusion systems – monitor the activity of a gene by fusing it to another
HeLa cells expressing gfp and rfp
Current favorites are the autofluorescent proteins
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
Restriction Enzyme Site Analysis
PCR Cloning –primer design Codon Usage Analysis Plasmid Construct – plasmid
drawing
PCR primer selection
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