1. 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

2. Host Organisms rDNA recipients Bacteria Yeast Mould Animal cell Plant cell

3. Transformation PlasmidCalcium chloride Heat shock Electroporation Phage/Cosmid/YACInfection

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

5. 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

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

7. gDNA library construction

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

9. gDNA library construction

11. cDNA library construction Isolation of poly (A) RNA

12. cDNA library construction cDNA preparation

13. Homopolymer tailing with TdT

14. cDNA library construction

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

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

17. Library screening

19. Chromosome walking

20. Differential hybridization Individual colonies Microarray Library screening

21. Differential hybridization

23. Subtractive Hybridization

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

25. 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

26. DDRT-PCR

30. 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