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  2. 2. Such manipulations of DNA are conducted by a toolkit of enzymes:  restriction endonucleases are used as molecular scissors,  DNA ligase functions to bond pieces of DNA together, and  a variety of additional enzymes that modify DNA are used to facilitate the process.
  3. 3. DNA modifying enzymes  Restriction enzymes and DNA ligases represent the cutting and joining functions in DNA manipulation.  All other enzymes involved in genetic engineering fall under the broad category of enzymes known as DNA modifying enzymes.  These enzymes are involved in the degradation, synthesis and alteration of the nucleic acids.
  4. 4. Nucleases  Nuclease enzymes degrade nucleic acids by breaking the phosphodiester bond that holds the nucleotides together.  Restriction enzymes are good examples of endonucleases, which cut within a DNA strand.  A second group of nucleases, which degrade DNA from the termini of the molecule, are known as exonucleases.
  5. 5. Nucleases and its action
  6. 6. Polymerases  Polymerase enzymes synthesise copies of nucleic acid molecules and are used in many genetic engineering procedures.  When describing a polymerase enzyme, the terms ‘DNA- dependent’ or ‘RNA-dependent’ may be used to indicate the type of nucleic acid template that the enzyme uses.  Thus, a  DNA-dependent DNA polymerase copies DNA into DNA,  an RNA-dependent DNA polymerase copies RNA into DNA, and  a DNA-dependent RNA polymerase transcribes DNA into RNA.
  7. 7. DNA Polymerases Mesophilic and thermophilic DNA polymerases for different polymerization reactions, DNA end blunting and amplification, labeling and others.  DNA Polymerase, Large Fragment  DNA Polymerase I  T4 DNA Polymerase  T7 DNA Polymerase  Terminal Transferase (TdT)
  8. 8. DNA Polymerase, Large Fragment •DNA Polymerase, Large Fragment, is a portion of DNA polymerase of Bacillus smithii, which catalyzes 5'=>3' synthesis of DNA and lacks 5'→3' and 3'→5' exonuclease activities. Highlights  Thermophilic DNA polymerase with strong strand displacement activity
  9. 9. DNA Polymerase I  DNA Polymerase I, a template-dependent DNA polymerase, catalyzes 5'→3' synthesis of DNA.  The enzyme also exhibits 3'→5' exonuclease (proofreading) activity, 5'→3' exonuclease activity, and ribonuclease H activity.
  10. 10. Highlights Incorporates modified nucleotides Active in multiple buffers, including restriction enzyme, PCR, and RT buffers Applications  DNA labeling  Second-strand synthesis of cDNA in conjunction with RNaseH
  11. 11. T4 DNA Polymerase  T4 DNA Polymerase, a template-dependent DNA polymerase, catalyzes 5'-3' synthesis from primed single-stranded DNA.  The enzyme has a 3'-5' exonuclease activity, but lacks 5'-3' exonuclease activity.
  12. 12. T7 DNA Polymerase  T7 DNA Polymerase, a template dependent DNA polymerase.  It catalyzes DNA synthesis in the 5'=>3' direction.  It is a highly processive DNA polymerase allowing continuous synthesis of long stretches of DNA.
  13. 13. Applications  Purification of covalently closed circular DNA by removal of residual genomic DNA  Primer extension reactions on long templates  DNA 3'-end labeling  Strand extensions in site-directed mutagenesis
  14. 14. Terminal Transferase (TdT)  Protruding, recessed or blunt ended double or single stranded DNA molecules serve as a substrate for TdT.  TdT is isolated and purified from an E. coli strain carrying the cloned terminal transferase gene from calf thymus.
  15. 15. DNA ligase  DNA ligase is an important cellular enzyme, as its function is to repair broken phosphodiester bonds that may occur at random or as a consequence of DNA replication or recombination.  It can therefore be thought of as molecular glue, which is used to stick pieces of DNA together.
  16. 16. Ligases  Fast and efficient ligation of DNA and RNA.  T4 DNA Ligase  T4 RNA Ligase
  17. 17. T4 DNA Ligase  The enzyme repairs single-strand nicks in duplex DNA, RNA, or DNA/RNA hybrids.  It also joins DNA fragments with either cohesive or blunt termini, but has no activity on single-stranded nucleic acids.  The T4 DNA Ligase requires ATP as a cofactor.
  18. 18. Applications  Cloning of restriction enzyme generated DNA fragments  Cloning of PCR products  Joining of double-stranded oligonucleotide linkers or adaptors to DNA  Site-directed mutagenesis
  19. 19. T4 RNA Ligase  T4 RNA Ligase catalyzes the ATP-dependent intra- and intermolecular formation of phosphodiester bonds between 5'-phosphate and 3'-hydroxyl termini of oligonucleotides, single-stranded RNA and DNA. Applications  Joining RNA to RNA  Specific modifications of tRNAs  Site-specific generation of composite primers for PCR
  20. 20. CONCLUSION:  These are the modifying enzymes represent the cutting and joining functions in DNA manipulation and genetic engineering.
  21. 21. REFERENCES

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