application in molecular cloning david shiuan department of life science, institute of biotechnology...

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