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Complementary DNA (cDNA) Libraries

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Complementary DNA (cDNA) Libraries

  1. 1. Speaker P.RAMESH Ph.D (ABC)
  2. 2. INTRODUCTION  In a simple cloning procedures, DNA fragments are joining to vector followed by introduction into the host cells & selection/screening will be done  If a source of a donor DNA is very complex??? Genomic DNA c DNA  Cloning strategy: DNA fragment Joining to Vector Host cell Selection/ screening
  3. 3.  Two major approaches for isolating sequences from complex sources (Genomic DNA/cDNAs) Cell based cloning strategy: It is to divide the source of DNA into fragments & clone everything Such a collection of clones representation of entire population called Library Screen the Library:To identify our clone of interest using a procedures that discriminates between the desired clone & all others
  4. 4. Cont… Target sequence by PCR: Selectively amplify the target sequences directly from source of DNA using PCR & cloned In PCR approach, screening step is built into 1st stage of the procedure So that only selected fragments are actually cloned
  5. 5. Complementary DNA (cDNA) Libraries  cDNA Library is a population of mRNAs  Generated by Reverse Transcription of cellular mRNA, reveals expression profiles in different cell types and developmental stages  Cloned eukaryotic cDNAs have their own special uses  Advantage: Size of cDNA clone is significantly lower than the Genomic DNA library
  6. 6. Cont…  Phage-λ vectors were mostly used for cDNA cloning & expression  λgt10 & λgt11 Phage vectors: Insertion Vectors Accept Approximately 7.6 kb & 7.2 kb  λZAP series: Phage clones have to be sub cloned back into plasmid called Phasmids Advantages: High capacity (up to 10Kb)
  7. 7. Early cDNA cloning Strategy 3 Major Steps: First-strand DNA synthesis on the mRNA by Reverse transcriptase Removal of mRNA template Second-strand DNA synthesis using 1st strand DNA as a template by DNA pol-I  cDNA cloning was based on the Homo-polymer tailing method (1970s)
  8. 8. Fig: An Early Cloning Strategy
  9. 9. Fig: Homo-polymer Tailing Method
  10. 10. Cont…  Disadvantage: Cleavage with S1 nuclease results in loss of certain amount of sequence at the 5’ end of the clone  Other Methods: Improved method by cDNA cloning (Land et al., 1981) Directional cloning Non-directional cloning (Gubbler-Hoffman method) CAPture method  Oligo-cappling method
  11. 11. Fig: Improved Method for cDNA cloning
  12. 12. Fig: Directional cDNA cloning method
  13. 13. Fig: Gubbler-Hoffman method
  14. 14. Cont…  Drawbacks: Generally 3’end bias in the resulting library Native enzymes have poor processivity & intrinsic RNase activity Optimal activity for native enzymes at 370C
  15. 15. Fig: CAPture Method for full length cDNA
  16. 16. Fig: Oligo-capping Method
  17. 17. Screening of cDNA Libraries Colony Hybridization N.Acids Hybridization Plaque-Lift Method Immunological Screening
  18. 18. Fig: Colony Hybridization Method
  19. 19. Fig: Plaqe lift Method
  20. 20. Fig: Immunological screeningMethod
  21. 21. Fig: Linear map of λZAP Phasmid vector

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