Methods for Plant Gene Cloning

1. Methods for Plant Gene Cloning • Heterologous PCR • Differential Hybridization • Reverse Genetics: Antibody, PCR w/guessmers • Gene tagging: promoter, inactivation, plasmid rescue

2. Methods for Plant Gene Cloning • Chromosomal walking: Genetic map:Physical map • EST: functional analysis w/ RNAi • Complementation: Yeast mutants • Two-hybrid system (BD+AD)

3. Plant Transformation Methods • Agrobacterium tumerfacience/rhizogenes • Particle bombardment • Microinjection: 침봉, 주사, 원형질체 • Electroporation

4. Library • Genomic Library: shortgun • cDNA Library: mRNA - Reverse Transcriptase – RNA/DNA hybrid – RNaseH - cDNA(complementary DNA) • 차이점

5. Library Screening • DNA probe • Oligonucleotide probes • Differential hybridization • Immuno-screening • Combinatorial screening

6. Vector: plasmids • Ds closed circular nonchromosoal DNA, 1-200Kbp • pBR322: Ampr, Tetr as selection markers • Multi-cloning sites • Replication origin; ColE1 • Promoters: T3, T7, Sp6 • Small size: easy transforamtion, handling, high copy number, less restriction site • β -galactosidase: insertional inactivation, X-gal(5-bromo-4-chloro-3-indolyl-β-D-galactoside)

7. Vector: Bacteriophage Lambda • 50Kbp ds DNA, 15Kb capacity • cloning only λgt10(6Kb) vs, expression λgt10(7.2Kb) • Lytic cycle vs. lysogenic cycle • Single Plaque: 106-107 pfu • 중앙의 1/3; lysis-unrelated region • 105-78% of WT can be packaged • Right arm + Left arm + insert • In vivo excision: λZap

8. Vector: Cosmid • 45Kbp capacity, 3X more than phage • “cos” containing plasmid • Distance between cos-cos: 45 Kbp for packaging

9. Vector: YAC (BAC, MAC) • Yeast artificial chromosome • 150Kbp • Centromere, 2 Telomeres, Origin of replication • Propagated as bacterial plasmid w/I bacteria • Behavior as chromosome w/I yeast