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

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

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

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

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

Vector: plasmids Ds closed circular nonchromosoal DNA, 1-200Kbp pBR322: Amp r, Tet r 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)

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

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

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