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
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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
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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
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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
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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
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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
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Informatics for Molecular Cloning
Restriction Enzyme Site Analysis
PCR Cloning –primer design Codon Usage Analysis Plasmid Construct – plasmid
drawing
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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
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Primer 3
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Codon Usage – differ from organisms
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