1. Application in Molecular Cloning David Shiuan Department of Life Science, Institute of Biotechnology and Interdisciplinary Program of Bioinformatics National Dong Hwa University

2. 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

3. 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 Plasmid Cloning Vector

13. 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

14. 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

15. 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

16. Informatics for Molecular Cloning Restriction Enzyme Site Analysis PCR Cloning – primer design Codon Usage Analysis Plasmid Construct – plasmid drawing

31. 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

33. Primer 3

36. Codon Usage – differ from organisms