Dna modification

1. UNIVERSITY INSTITUTE OF BIOTECHNOLOGY
BIOTECHNOLOGY
Bachelor's of Science
B.Sc. Biotechnology/ III Sem
Genetic Engineering
(24BTT-216)
Unit No. 1 Chapter No. 1 Lecture No. 1.2.1
Topic : DNA modifying enzymes
Dr. Anil Kumar (E8226) Designation: Associate Professor
Academic Session 2025-26
ODD Semester Jul-Dec 2025

2. Learning Objectives
1. After completion of the course the students will be
acquainted with the basics of Genetic engineering and
cloning vectors
2. After completion student will learn about the role of
restriction endonucleases in genetic engineering
3. After completion student will learn about the role and
function of end modifying enzymes.
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3. Recap of Previous Lecture
2
Last lecture we have studied about
History of genetic engineering
Different discoveries related to the rise of
genetic engineering
Role and importance of enzyme that cut DNA
at specific sites

4. Applications of the Topic
• Insight about the cutting of
DNA with restriction
endonucleases
• Mechanism of action of
endonucleases
• Why Host DNA is unaffected
• How DNA can be modified at
ends
Figure-2 : Supportive image / video / chart /
graphics with source reference (as applicable)
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5. Summary of the Lecture
1. Genetic engineering refers to the direct manipulation of
an organism’s genome using advanced DNA technology.
2. It involves the introduction, deletion or modification of
genes within an organism’s DNA to produce desirable
traits.
3. Genetic engineering has revolutionised fields like
agriculture, medicine and biotechnology enabling
innovations like disease-resistant crops, synthetic insulin
production and gene therapy....
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6. Introduction
• To produce recombinant molecule:
the vector
the DNA to be cloned
cut at specific points and then joined
together in a controlled manner.

7. DNA manipulative enzymes
• Nucleases are enzymes that cut, shorten, or degrade nucleic acid
molecules.
• Ligases join nucleic acid molecules together.
• Polymerases make copies of molecules.
• Modifying enzymes remove or add chemical groups.

9. Nucleases
• Nucleases degrade DNA molecules by breaking the phosphodiester
bonds that link one nucleotide to the next in a DNA strand. There are
two different kinds of nuclease
• Exonucleases remove nucleotides one at a time from the end of a
DNA molecule.
• Endonucleases are able to break internal phosphodiester bonds
within a DNA molecule

10. Pic source: Brown, T.A., 2016. Gene cloning and DNA analysis: an introduction. John
Wiley & Sons.

11. Ligase
• In the cell the function of DNA ligase is to repair
single-stranded breaks.
• DNA ligases from most organisms can also join
together two individual fragments of DNA.
Pic source: Brown, T.A., 2016. Gene cloning and DNA analysis: an introduction. John
Wiley & Sons.

13. DNA polymerases
DNA polymerase I E. coli. attaches to a short single-stranded region (or
nick) in a mainly double-stranded DNA
molecule, and then synthesizes a completely
new strand.
DNA polymerization and DNA degradation.
subtilisin cleaved to large and small subunit .
The larger subunit known as Klenow fragment.
Taq DNA
polymerase
Thermus
aquaticus.
polymerase chain reaction (PCR) Remain active at 94°C .
Reverse
transcriptase
Retro-viruses Reverse
transcriptase is unique in that it uses as a
template not DNA but RNA
The ability of this enzyme to synthesize a DNA
strand complementary to an RNA template
is central to the technique called
complementary DNA (cDNA) cloning

14. • When E. coli DNA polymerase 1 is proteolytically digested by the bacterial
protease subtilisin, it produces two fragments – a large fragment and a small
fragment.
• The Klenow fragment is the largest fragment that contains 5 to 3 polymerase
′ ′
and 3 to 5 exonuclease (proofreading) activity domains of the DNA
′ ′
polymerase .
Ⅰ
• The Klenow fragment’s 3 to 5 exonuclease activity aids in the elimination of
′ ′
improperly inserted bases while polymerisation proceeds.
• It lacks the 5 to 3 exonuclease activity, which is shown by full-length or entire
′ ′
E. coli DNA polymerase .
Ⅰ
Klenow Fragment

17. The Klenow fragment is extremely useful for research-based tasks such
as: Synthesis of double-stranded DNA from single-stranded templates.
Filling in receded 3' ends of DNA fragments to make 5' overhang blunt.
Digesting away protruding 3' overhangs.

18. Reverse Transcriptase

19. DNA modifying enzymes
• There are numerous enzymes that modify DNA molecules
by addition or removal of specific chemical groups.
• The most important are as follows:
• Alkaline phosphatase (from E. coli, calf intestinal tissue, or
arctic shrimp), which removes the phosphate group
present at the 5 terminus of a DNA molecule.
′
Pic source: Brown, T.A., 2016. Gene cloning and DNA analysis: an introduction. John Wiley
& Sons.

20. • Polynucleotide kinase (from E. coli infected with T4
phage), which has the reverse effect to alkaline
phosphatase, adding phosphate groups onto free 5′
termini.
Pic source: Brown, T.A., 2016. Gene cloning and DNA analysis: an introduction. John
Wiley & Sons.

21. • Terminal deoxynucleotidyl transferase (from calf
thymus tissue), which adds one or more
deoxyribonucleotides onto the 3 terminus of a
′
DNA molecule.
Pic source: Brown, T.A., 2016. Gene cloning and DNA analysis: an introduction.
John Wiley & Sons.

22. Next Lecture
Topic(s)
 Discuss about the role of restriction endonucleases in
cutting of DNA at specific sites
 What are recognition sites
 Different enzymes used in modification of DNA at specific
sites
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23. Quiz/ FAQ’s
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1. Cutting and joining of the DNA are which techniques?
a) DNA degradation
b) DNA replication
c) DNA manipulation
d) DNA synthesis
View Answer
2. What type of DNA enzymes is made use of in most of the DNA manipulative
techniques?
a) Partially degraded
b) Purified
c) Degraded or denatured
d) Enclosed in a parent cell
View Answer
3. Enzymes that remove nucleotides one at a time from the end of a DNA molecule are
called
a) Ligases
b) Exonucleases
c) Endonucleases
d) Modifying enzymes

24. References/ Articles/ Videos
9
GENE CLONING AND DNA ANALYSIS: AN INTRODUCTION,
8TH EDITION Paperback – 1
0 December 2020, T. A. Brown
GENE CLONING AND DNA ANALYSIS: AN INTRODUCTION, 8TH EDITION Paperback – 10 December 2020,
T. A. Brown
References
1. Buckley, G. (15 Janurary 2021). Nucleic Acid. Retrieved 07 May, 2023,
from https://biologydictionary.net/nucleic-acid/
2. Verma, P. S., & Agrawal, V. K. (2006). Cell Biology, Genetics, Molecular Biology, Evolution &
Ecology (1 ed.). S .Chand and company Ltd.
3. Klug, W. S., & Cummings, M. R. (2003). Concepts of genetics. Upper Saddle River, N.J: Prentice
Hall.
4. https://www.britannica.com/science/restriction-enzyme
5. https://international.neb.com/products/restriction-endonucleases/restriction-endonucleases
6. https://en.wikipedia.org/wiki/Restriction_enzyme
7. http://www.bio.miami.edu/dana/dox/restrictionenzymes.html
8. Chen, K., Zhao, B. S., & He, C. (2016). Nucleic acid modifications in regulation of gene expression.
Cell chemical biology, 23(1), 74-85.

25. References
• Brown, T.A., 2016. Gene cloning and DNA analysis:
an introduction. John Wiley & Sons.
• An Introduction to Genetic Engineering- Desmond
S. T Nicholl
• Gene Manipulation: Old and Primrose, 2000.

26. Faculty-curated videos, NPTEL,
Coursera, LinkedIn, or other relevant
learning resources
https://youtu.be/4OGAz1j5yF8?si=Vy_q4ZFFjbKB3xyc
https://youtu.be/ixnVL-m0-QA?si=02PnVG9kHbBmjpwA
https://archive.nptel.ac.in/courses/105/104/105104183/
https://youtu.be/2pp17E4E-O8?si=t4H0uncCPc1xpHCp
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27. Class-Wise Feedback
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Scan QR code for your experience regarding the lecture

28. Thank You
For queries
Email: anil.e8226@cumail.in
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