Important Restriction Enzymes in Molecular Biology

1. Molecular Biotechnology
Restriction Enzymes
Vaibhav Kulkarni
Saraswathi Vidya Bhavan’s College of Pharmacy

3. Reverse Transcriptase
Reverse transcriptase is an enzyme that
makes a DNA strand using mRNA as a
template strand .it is also known as RNA
Dependent DNA synthetase.
It is isolated from retroviruses[Rous
Sarcoma Virus (RSV ) , Mouse Mammary
tumor virus (MMTV), Murine leukaemia
Virus (MuLV)]

4.  The enzyme is bound with RNA core of the
Viruses. It is 70,000 Daltons ( 70 k Da)
in molecular weight .
It adds complementary Deoxyibonucleotides on
mRNA template to synthesize complementary
DNA (c-DNA).
The enzyme requires free 3’ OH group to add first
complementary base to the mRNA . 3’ OH
group is provided by the primer.
Use: Reverse transcriptase is used to make
c-DNA from m-RNA

6. Restriction Endonucleases
Also called restriction enzymes
1962: “molecular scissors” discovered in in bacteria
E. coli bacteria have an enzymatic immune system that
recognizes and destroys foreign DNA
3,000 enzymes have been identified, around 200 have
unique properties, many are purified and available
commercially

7.  The nuclease enzyme that cuts the DNA
at unique sequence is called as restriction
endonuclease.
 They cut the DNA in non terminal region .
restriction endonucleases are used to
generate rejoinable DNA Fragments.
 Molecular scissors
 Molecular knives
 Molecular scalpels

8.  The sequence recognized by RE to cut the
DNA is called
restriction site ,
restriction endonulcease site ,
Recognition site .

9. Mechanism
 The RE breaks two phosphodiester bonds,
One in either strand of the duplex DNA to cut the
DNA .
The 3’ cut end has free – OH group .
The 5’ cut end has a phosphate group .
The genome of an organism has several restriction
site for one RE.
The distance between two adjacent restriction site
varies greatly .so RE produces several DNA
Fragments of different lengths while cutting DNA

10. Restriction Endonucleases
Named for bacterial genus, species, strain, and type
Example: EcoR1
Genus: Escherichia
Species: coli
Strain: R
Order discovered: 1

11. Restriction Endonucleases
Recognition sites have symmetry (palindromic)
“Able was I, ere, I saw Elba”
Bam H1 site:
5’-GGATCC-3’
3’-CCTAGG-5’

12. Restriction Endonucleases
Enzymes recognize specific 4-8 bp sequences
Some enzymes cut in a staggered fashion - “sticky ends”
EcoRI 5’…GAATTC…3’
3’…CTTAAG…5’
Some enzymes cut in a direct fashion – “blunt ends”
PvuII 5’…CAGCTG…3’
3’…GTCGAC…5’

14. Restriction Endonucleases
Why don’t bacteria destroy their own
DNA with their restriction enzymes?

15. Methylation

16. Uses for Restriction Enzymes
RFLP analysis (Restriction Fragment Length Polymorphism)
DNA sequencing
DNA storage – libraries
Transformation
Large scale analysis – gene chips

17. Human DNA cleaved with EcoRI Corn DNA cleaved with EcoRI
5’-C-G-G-T-A-C-T-A-G-OH
3’-G-C-C-A-T-G-A-T-C-T-T-A-A-PO4
PO4-A-A-T-T-C-A-G-C-T-A-C-G-3’
HO-G-T-C-G-A-T-G-C-5’+
5’-A-C-G-G-T-A-C-T-A-G A-A-T-T-C-A-G-C-T-A-C-G-3’
3’-T-G-C-C-A-T-G-A-T-C-T-T-A-A G-T-C-G-A-T-G-C-5’
Complementary base pairing
+ DNA Ligase, + rATP
recombinant DNA molecule
5’-A-C-G-G-T-A-C-T-A-G-A-A-T-T-C-A-G-C-T-A-C-G-3’
3’-T-G-C-C-A-T-G-A-T-C-T-T-A-A-G-T-C-G-A-T-G-C-5’
Restriction Enzymes for Transformation

18. _
+
DNA is negatively
charged from the
phosphate backbone
Visualize DNA with ethidium
bromide – fluoresces ONLY
when bound to DNA
Restriction Enzymes for RFLP

19. DNA Bands with Restriction Enzymes

20. Blunt Ends of DNA

21. Cohesive ends of DNA

22. Examples of RE

23. RE Reaction

24. DNA Ligase
 DNA ligase is a specific type of enzyme, a ligase, that
facilitates the joining of DNA strands together by
catalyzing the formation of a phosphodiester bond. It
plays a role in repairing single-strand breaks in
duplex DNA in living organisms, but some forms (such
as DNA ligase IV) may specifically repair double-strand
breaks (i.e. a break in both complementary strands of
DNA). Single-strand breaks are repaired by DNA ligase
using the complementary strand of the double helix as a
template,[1] with DNA ligase creating the final
phosphodiester bond to fully repair the DNA.

25.  DNA ligase has applications in both DNA
repair and DNA replication ( Mammalian
ligases). In addition, DNA ligase has extensive
use in molecular biology laboratories
for recombinant DNA experiments ( Applications
in molecular biology research). Purified DNA
ligase is used in gene cloning to join DNA
molecules together to form recombinant DNA.

26. DNA ligase repairing
chromosomal damage

27. DNA Ligase

28. Uses1. To join a vector DNA and a target DNA to
Construct r- DNA .
2.It is used to join DNA fragments of
different organisms for making vectors
with desired characters .
3.Used to add linker and adaptor sequences
to blunt ended vector DNA and Target
DNA.
4.It is used to join oligonucleotides together
in the chemical synthesis of DNA by ligase
chain reaction( LCR).