DNA cloning General strategies Choose DNA sources (gDNA/cDNA) Produce collection of DNA fragments Join them to appropriate vector Introduce rDNA to a host cell Screen/Select transformants

Host Organisms rDNA recipients Bacteria Yeast Mould Animal cell Plant cell

Transformation PlasmidCalcium chloride Heat shock Electroporation Phage/Cosmid/YACInfection

DNA library Collection of cloned DNA sequences in host cells Require complete or near complete representatives of genome

DNA library Large DNA fragments  Fewer number of clones Library of genome with 3 * 10 9 bp Inserts of 20 kb long Require 1.5 * 10 5 recombinant molecules

Types of DNA library Genomic Library representing the entire genome cDNA Library representing only expressed genome

gDNA library construction

Partial digestion of genomic DNA Ligation of fragments to vector of choice: plasmid, phage, cosmid, BAC or YAC

gDNA library construction

cDNA library construction Isolation of poly (A) RNA

cDNA library construction cDNA preparation

Homopolymer tailing with TdT

cDNA library construction

Library storage Homogeneous aliquots Deep freeze at -70 / -80 Celcius 20 % glycerol 7% DMSO

Library screening Southern/Western hybridization using specific probe Chromosome walking Differential hybridization Subtractive hybridization

Library screening

Chromosome walking

Differential hybridization Individual colonies Microarray Library screening

Differential hybridization

Subtractive Hybridization

DDRT-PCR Differential Display Reverse transcription (ase) Polymerase Chain Reaction Screening of differentially expressed genes Without library

DDRT-PCR Subsets of mRNA to be amplified Using 3’ oligo d(T) + dinucleotide primers 5’ arbitrary primers Size fractionation with PAGE Identification of polymorphic bands Sequencing and Expression verification

DDRT-PCR

Genomic / cDNA clone Identification:Genome organization (gene/nongene) Gene structure (exon/intron) Regulatory regions/sequences Induction of mutation for functional analysis Identification:Differentially expressed genes Expression profiles