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DNA cloning

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.

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DNA cloning

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  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 Plasmid Calcium chloride Heat shock Electroporation Phage/Cosmid/YAC Infection

  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 * 109 bp Inserts of 20 kb long Require 1.5 * 105 recombinant molecules

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

  7. gDNA library construction

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

  9. gDNA library construction

  10. 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

  18. Library screening

  19. Chromosome walking

  20. Differential hybridization Individual colonies Microarray Library screening

  21. Differential hybridization

  22. 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

  27. DDRT-PCR

  28. DDRT-PCR

  29. 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

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