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
Plasmid
Calcium chloride
Heat shock
Electroporation
Phage/Cosmid/YAC
Infection
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 * 109 bp
Inserts of 20 kb long
Require 1.5 * 105 recombinant molecules
Types of DNA library
Genomic Library
representing the entire genome
cDNA Library
representing only expressed genome
gDNA library construction
gDNA library construction
Partial digestion of genomic DNA
Ligation of fragments to vector of choice:
plasmid, phage, cosmid, BAC or YAC
gDNA library construction
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
Library screening
Chromosome walking
Differential hybridization
Individual colonies
Microarray
Library screening
Differential hybridization
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
DDRT-PCR
DDRT-PCR
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