1. DNA manipulative enzymes
Dr. Mayank Chaudhary
Assistant Professor
Department of Biotechnology
Maharishi Markandeshwar (Deemed to be University)
Mullana-Ambala, Haryana
2. • Classification based on type of reaction catalyzed:
1) Nucleases: Cut/degrade nucleic acids
2) Ligases: Join nucleic acid molecules
3) Polymerases: Make copies of molecules
4) Modifying enzymes: Remove/add chemical groups
3. Nucleases
• Degrade DNA molecules by
breaking phosphodiester bonds
that link one nucleotide to the
next in a DNA strand. Types:
1) Exonuclease: Remove
nucleotides from the end of DNA
molecule.
2) Endonuclease: Breaks internal
phosphodiester bonds within
DNA molecule.
4.
5.
6.
7. Ligases
• Repair single-stranded breaks
that arise in double stranded
DNA molecules during
replication.
• Can also join two individual
fragments of double stranded
DNA.
• Catalyzes covalent closure of
nicks (missing phosphodiester
linkages, no missing bases) by
using energy from NAD or
ATP
8. Polymerases
• Enzymes that synthesize new strand of DNA complementary to an existing
DNA or RNA template. Types:
• DNA pol I: Isolated from E.coli and attaches to short, single stranded
region mainly in double stranded DNA molecule. It then synthesizes
complementary new strand degrading existing strand as it proceeds.
• Klenow fragment: Modified DNA pol I that retains polymerase activity but
no nuclease activity due to removal of segment.
• DNA pol I from bacterium Thermus aquaticus is the Taq DNA polymerase
used in PCR.
• Reverse Transcriptase: Uses RNA as template for synthesis of cDNA.
9.
10. DNA modifying Enzymes
1) Alkaline Phosphatase: Removes phosphate group present at 5’ end
of DNA molecule.
2) Polynucleotide Kinase: Adds phosphate group onto 5’ end.
3) Terminal deoxynucleotidyl transferase: Adds one or more dNTPs
onto 3’ end of DNA molecule.
11.
12. Restriction Endonucleases (REs)
• Discovered in strains of bacteria that were immune to bacteriophage
infection due to phenomenon called as Host-controlled restriction.
• First RE to be discovered: HindII by Nathans, Arber and Smith.
13. • Nomenclature:
Classification of REs:
Type I: Possess both restriction and modification activity. Cleavage occurs 1000bp
away from recognition site.
Type II: Restriction and modification activities are mediated by separate enzymes.
Recognizes palindromic sequence and cleavage occurs at restriction site.
Type III: Recognition and methylation of DNA sequence and cleavage at 24-26bp
away.
14. Number of restriction sites/recognition sequences for particular RE in a DNA
molecule: 4n (n= length of recognition sequence)
Eg. For GATC recognition sequence: 44=256 nucleotides
For GGATTC recognition sequence: 46=4096 nucleotides
15. • Blunt ends/flush ends: Double stranded cut in the middle of recognition
sequence. Eg. PvuII and AluI are blunt end cutters.
• Sticky/cohesive ends: Two DNA strands are not cut at exactly same
position and cleavage is staggered/ Eg. NotI and EcoRI.
It is easier for DNA ligase to work on
sticky ends due to hydrogen bonding
between complementary bases.
16. How Blunt ends can be converted to Sticky
ends??
1) Linkers
2) Adaptors
3) Homopolymer tailing
Linkers: Blunt-ended double stranded DNA molecules containing restriction site.
17. Adaptors: Short synthetic oligonucleotide similar to linkers but is pre-treated to
have one sticky end.
18. Homopolymer tailing: Series of nucleotides is attached to 3’-OH termini of double
stranded DNA molecule with help of enzyme terminal deoxynucleotidyl transferase.
19. References
• T. A. Brown. Gene cloning & DNA analysis-An Introduction.
Seventh edition.
