RECOMBINANT DNA TECHNOLOGY

1. JIWAJI UNIVERSITY
CENTER FOR GENOMICS
MOLECULAR & HUMAN GENETICS
Presented by:-
AKANSHA CHAUHAN
3rd SEMESTER

3. CONTENTS
 Introduction
 Principle of rDNA technology
 Applications of rDNA technology
 Restriction Endonucleases enzyme
 DNA Ligase
 DNA Polymerase
 DNA Modifying enzymes

4. Introduction
 Recombinant DNA Technology is the joining together of
DNA molecules from two different species.
 The Recombinant DNA molecule is inserted into a host
organism to produce new genetic combinations.
 Recombinant DNA technology also known as Gene
Cloning/Molecular Cloning, Genetic Engineering, Genetic
modification.
 The first recombinant molecule containing DNA from
different organisms was assembled late in 1971.
 The technology for propagating and expressing
recombinant genes was invented by Stanley Cohen and
Herbert Boyer in 1973.

5. Basic principles of rDNA
technology
 Generation of DNA fragments & selection of the desired
piece of DNA.
 Insertion of the selected DNA into a cloning vector to create
rDNA.
 Introduction of the recombinant vectors into host cells.
 Multiplication & selection of clones containing the
recombinant molecules.
 Expression of the gene to produce the desired product.

6. Recombinant DNA Technology
Britanica.com

7. Applications of Recombinant DNA
Technology
 rDNA technology has a wide range of application in
industries, medical science, & agriculture as “well as
molecular biology”.
 Molecular diagnosis of diseases
 Gene therapy
 DNA fingerprinting
 Production of vaccines
 Commercial & pharmaceutical products.

8. Enzymes used for rDNA
Technology
 rDNA technology produce rDNA using a set of enzymes
known as recombinant enzymes.
 They are :-
1. Nucleases
2. Ligases
3. Polymerases
4. DNA modifying enzymes

9. Restriction Endonucleases
Enzymes
 Restriction endonucleases are indispensable for DNA cloning &
sequencing. They serve as the tools for cutting DNA molecules
at predetermined sites, which is the basic requirement for gene
cloning or rDNA technology.
 The presence of restriction enzymes was postulated by W.
Arber, during 1960s, while the first true restriction
endonuclease was isolated in 1970 by Smith, Nathans.
 Nucleases are enzymes that degrade DNA molecules by
breaking the phosphodiester bonds.
 Nucleases are two types:-
 Exonuclease : removes the terminal nucleotide of the DNA
molecule

10. Continued
 Endonucleases : breaks the internal phosphodiester bond.
 Endonucleases are the most widely used ones.
 Restriction Endonucleases were first discovered in E.coli.
 There are four distinct types of restriction endonucleases:
i. Type I
ii. Type II
iii. Type III
iv. Type IV

11. Feature Type I Type II Type III Type IV
Structural
subunits
Three different Two identical Two different Two
different
Enzyme
activity
Endonuclease,
Methyltransferas
e ATPase
Endonuclease or
methyltransferase
Endonuclease,
methyltransferase
ATPase
Endonuclea
se GTPase
Biochemical
cofactors for
DNA
cleavage
ATP, AdoMet,
Mg2+
Mg2+ ATP, Mg2+ Mg2+, GTP
Methylation AdoMet, Mg2+ AdoMet AdoMet, Mg2+ —
Recognition
sequence
Asymmetric,
bipartile
Usually symmetric Asymmetric Bipartile,
methylated
Cleavage site Random, atleast
1000bp from
recognition site
At or near
recognition site
25-27bp from
recognition site
Between
methylated
bases at
multiple
positions
DNA
translocation
Yes No Yes Yes

12. Nomenclature
 The nomenclature of restriction endonucleases follows a
general pattern.
 The 1st letter of the name of genus in which a given enzyme
is discovered is written in capital.
 This is followed by the first two letters of species name of
the organism.
 These three letters are written in italics, as Eco from
Escherichia coli, Hin from Haemophilus influenzae.
EcoRI :- E = genus, co = species, R = strain(Ry13), I= order of
discovery Ist .

13. Recognition Sequence
 It is a site where DNA is cut by Restriction endonuclease.
 It is 4-8bp in sequence.
 It is pallindromic in sequence. Example- ROTATOR
5’GAATTC3’
3’CTTAAG5’
 Most Type II restriction endonucleases have recognition sites of 4, 5,
or 6bp, which are GC – rich.
 EcoRI recognition site
5’GAA TTC3’
3’CTT AAG5’

14. Mode of action of Restriction
Enzyme
 Restriction enzymes cuts DNA molecules in two different
styles.
 Sticky or Staggered style
• Most common cutting by Restriction Endonucleases
• Complementary ends present.
• Examples- EcoRI, BamHI
EcoRI - 5’GAATTC3’ 5’GAATTC3’
3’CTTAAG5’ 3’CTTAAG5’
BamHI - 5’GGATCC3’ 5’GGATCC3’
3’CCTAGG5’ 3’CCTAGG5’

15. Continued
 Blunt Style
• Less common used in RDT
• No complementary ends present.
• Example HaeIII, AluI
HaeIII- 5’GG CC3’
3’CC GG5’
AluI - 5’AG CT3’
3’TC GA5’

16. DNA Ligase
 DNA ligase act as a molecular glues as they help in sealing
gaps in DNA fragments.
 In 1969 Har Govind Khorana discovered DNA ligase in T4
bacteriophage.
 The enzyme used most often in RDT is T4DNA ligase.
 DNA ligase joins two ends of DNA & help in the synthesis of
phosphodiester bond. This phosphodiester bond is formed
between 3’OH group at the end of 1 polynucleotide chain &
5’phosphate group at the end of other polynucleotide chain
of DNA.
 This enzyme changes linear DNA into circular DNA & helps
in formation of recombinant molecule.

17. Continued
 Restriction digestion of DNA generates two types of DNA
ends- sticky ends or blunt ends.
 Blunt ends are simple direct and non-cohesive.
 Special types of DNA ligase are used to ligate these types of
DNA ends.
 Sticky ends are cohesive & have few basepairs palindromic
sequences on both the strands.
 Sticky ends are generated for inserting a gene of interest in
the plasmid.
 It works better in comparison with the blunt end ligation.

18. DNA Ligase
Sticky end Blunt end
researchgate.co geneticeducation.co.in

19. DNA Polymerase
 DNA polymerase is a complex enzyme which synthesize
nucleotide complementary to template strand.
 It adds nucleotide to free 3’OH end and help in elongation
of strand.
 It also helps to fill gap in double stranded DNA.
 DNA polymerase was first discovered by Kornburg in 1959 in
E.coli bacteria.
 Taq polymerase isolated from Thermus aquaticus is used in PCR.
 There are three types of DNA polymerases:-PolymeraseI,
PolymeraseII, DNA polymeraseIII.

20. continued
 The DNA polymeraseI enzyme, apart from addition of single
nucleotide to free OH end of DNA chain, also catalyzes 3’ 5’
& 5’ 3’ exonuclease activity.
 DNA polymerasesII also catalyzes 3’ 5’ exonucleases
activity.
 The polymerization activity of polymeraseII is less than the
polymeraseI & about 50 nucleotides per minute are synthesized.
 DNA polymerasesIII requires an auxiliary protein DNA
copolymerase III for its polymerization activity. It can polymerize
15000 nucleotide per minute.

21. DNA Polymerase
chromoscience

22. DNA modifying enzymes
 There are numerous enzymes that modify DNA molecules
by addition or removal of specific chemical groups.
 The most important are as:-
 Alkaline phosphatase
 Polynucleotide kinase
 Terminal deoxynucleotidyl transferases

23. Alkaline phosphatase
• It is isolated from E.coli or calf intestinal tissue.
• It removes the phosphate group present at 5’terminal of the
DNA molecule.
• This enzyme is used to stop the ligation reaction between
the DNA molecules.
biologyexams4u.com

24. Polynucleotide kinase
 It isolated from E.coli which has reverse effect of alkaline
phosphates add ℗ terminal in the end.
 It uses ATP as phosphate donor in reaction
 It promotes ligation reaction
brainkart.com

25. Terminal deoxynucleotidyl
transferases
 This enzyme is responsible for Homopolymer tailing.
 This enzyme involves the addition of oligo to 5’ends of DNA
& addition of oligo to 3’end of DNA.
 The complementary end of DNA can be joined by annealing
process.
 The tailing is catalyzed by enzyme (ToT)
biologyexams4u.com

26. Conclusion
 Recombinant DNA technology is an important development
in science that has made the human life much easier. It has
advanced strategies for cancer treatment, genetic diseases,
diabetes.
 This technology also involved in forensic science.
 The combination of restriction modification enzymes
representing cutting and joining functions in DNA
manipulation and genetic engeeniring enable the
application of a wide variety of procedures.

27. References
 nptel.ac.in
 onlinebiologynotes.com
 sciencedirect.com
 prezi.com
 britannica.com
 biologynotes4u.com
 brainkart.com