the Secrets of DNA Manipulation: A Comprehensive Exploration of DNA Polymerase and Enzymes In this PDF presentation entitled "Enzymes that Manipulate DNA, Specially DNA Polymerase," we delve deep into the mechanisms and functions of these remarkable enzymes that play a pivotal role in the realm of molecular biology. 🧬 Key Highlights: Introduction to DNA Polymerase: Uncover the fundamental aspects of DNA polymerase, a key player in DNA replication and repair. Explore its structure, functions, and the indispensable role it plays in maintaining the genetic integrity of living organisms. Types of DNA Polymerases: Delve into the diverse landscape of DNA polymerases, ranging from prokaryotic to eukaryotic systems. Understand how different types of DNA polymerases contribute to the precision and efficiency of DNA synthesis. Examples of polymerases: •DNA polymerase 1 •klenow fragment •sequenase •Taq polymerase •Reverse Transcriptase DNA Replication Take a closer look at the intricate dance of enzymes during DNA replication. Follow the step-by-step process, and gain insights into how DNA polymerase ensures the accurate transmission of genetic information from one generation to the next. Technological Applications: Unleash the potential of DNA polymerase in various biotechnological applications. From PCR (Polymerase Chain Reaction) to DNA sequencing, discover how these enzymes have revolutionized molecular biology and genetic research. Emerging Trends and Future Prospects: Stay ahead of the curve by exploring the latest advancements and emerging trends in DNA manipulation. Witness the ongoing research that promises to unlock new possibilities in the field. 🎓 Who Should Explore This Presentation? Students and researchers in molecular biology and genetics Biotechnologists and professionals in the field of genetic engineering Enthusiasts curious about the molecular machinery behind DNA manipulation

1. DNA manipulation
Enzymes

2. DNA Polymerase
template dependent
•DNA dependent DNA
Polymerase
➢DNA polymerase 1
➢Klenow fragments
➢Sequenase
➢Taq polymerase
•RNA dependent DNA
Polymerase
➢Reverse transcriptase
Nucleases
•Exonucleases
•Bal31
•Exonuclease III
•Endonucleases
•DNSI
•S1 Nuclease
•Restriction endonuclease
Ligases
•T4 DNA ligase
•T4 RNA ligase
End-modification
enzymes
The enzymes used for DNA manipulation
fall into 4 main categories

3. DNA Polymerases
• Enzyme that synthesizes DNA.
• An enzyme that copies an existing DNA or RNA molecule is called a
template – dependent DNA polymerase.
• Many of the techniques used to study DNA depend on the synthesis
of copies of all or part of existing DNA or RNA molecules such as:
1. PCR
2. DNA sequencing
3. DNA labelling

4. Mode of Action of a Template-
dependent DNA Polymerase
1. A template – dependent DNA polymerase makes a new DNA
polynucleotide whose sequence is dictated via→ the base pairing
rules.
• synthesized in the 5' to 3' direction.
• To initiate DNA synthesis there must be a primer (short double –
stranded region) ??G.R
➢ to provide a 3 ' end onto which the enzyme will add new
nucleotides.

5. Many of the template - dependent DNA
polymerases are multifunctional
• being able to
➢degrade DNA molecules
➢synthesize DNA molceules (5'-3 ' synthesis capabilities)
➢they can have one or both of the following exonuclease activities
1. 3 ' -5 ' exonuclease activity (remove nucleotides from the 3 ' end )
Proof-reading activity.
2. 5 '- 3 ‘ exonuclease activity

7. DNA polymerase 1
• A single polypeptide chain ( Mr 109 000 d)
• Enzymatic activity:
1. 5' to 3' polymerase (synthesis)
2. 5' to 3’ exonuclease activity.
3. 3' to 5' exonuclease activity.

8. Uses

9. 1)Labeling DNA by nick translation
• Its one of the oldest probe labeling techniques
• Once the DNA has been labeled it can be used:
➢as a probe molecular hybridization experiments (used to detect
complementary sequences on Southern blots)
➢in recombinant organisms by colony or plaque hybridization
➢provides a means of detecting picogram amounts of DNA on a gel by
autoradiography, whereas ethidium bromide staining will only detect
about 50-100 ng of DNA per band.

10. Dnase → nick the single strand at the
phosphodiester backbone

11. Dnase → nick the single strand at the
phosphodiester backbone
DNA Pol I → degrade the non template strand by
5’ to 3’ exonuclease activity

12. DNA Pol I → degrade the non template strand by
5’ to 3’ exonuclease activity
Dnase → nick the single strand at the
phosphodiester backbone
Synthesize copy template by α-^33P [dNTP]

13. Dnase → nick the single strand at the
phosphodiester backbone
DNA Pol I → degrade the non template strand by
5’ to 3’ exonuclease activity
DNA Pol I → Synthesize copy template by α-
^33P [dNTP]

14. DNA Pol I → Synthesize copy template by α-
^33P [dNTP]
DNA Pol I → degrade the non template strand by
5’ to 3’ exonuclease activity
Dnase → nick the single strand at the
phosphodiester backbone

15. Dnase → nick the single strand at the
phosphodiester backbone
DNA Pol I → degrade the non template strand by
5’ to 3’ exonuclease activity
DNA Pol I → Synthesize copy template by α-
^33P [dNTP]

16. 2) DNA Replication

17. 3) Proof-reading (correcting mis-
matched nucleotides

18. 4)cDNA cloning 2nd strand synthesis
Starting material:
RNA (mRNA) + Poly (A)

19. 4)cDNA cloning 2nd strand synthesis
Starting material:
RNA (mRNA) + Poly (A)
Short oligo(dT) primer is annealed the
poly (A) tail on the mRNA provides the
3’ OH for RT to begin coping the mRNA

20. 4)cDNA cloning 2nd strand synthesis
Starting material:
RNA (mRNA) + Poly (A)
Short oligo(dT) primer is annealed the
poly (A) tail on the mRNA provides the
3’ OH for RT to begin coping the mRNA

21. 4)cDNA cloning 2nd strand synthesis
Starting material:
RNA (mRNA) + Poly (A)
Short oligo(dT) primer is annealed the
poly (A) tail on the mRNA provides the
3’ OH for RT to begin coping the mRNA

22. 4)cDNA cloning 2nd strand synthesis
Starting material:
RNA (mRNA) + Poly (A)
Short oligo(dT) primer is annealed the
poly (A) tail on the mRNA provides the
3’ OH for RT to begin coping the mRNA
mRNA is removed by Rnase H

23. 4)cDNA cloning 2nd strand synthesis
Starting material:
RNA (mRNA) + Poly (A)
Short oligo(dT) primer is annealed the
poly (A) tail on the mRNA provides the
3’ OH for RT to begin coping the mRNA
mRNA is removed by Rnase H
Nicks are created in the mRNA strand

24. 4)cDNA cloning 2nd strand synthesis
Starting material:
RNA (mRNA) + Poly (A)
Short oligo(dT) primer is annealed the
poly (A) tail on the mRNA provides the
3’ OH for RT to begin coping the mRNA
mRNA is removed by Rnase H
Nicks are created in the mRNA strand
With DNA polymerase I→ a nick translation
reaction synthesize the second cDNA strand

25. 4)cDNA cloning 2nd strand synthesis
Starting material:
RNA (mRNA) + Poly (A)
Short oligo(dT) primer is annealed the
poly (A) tail on the mRNA provides the
3’ OH for RT to begin coping the mRNA
mRNA is removed by Rnase H
Nicks are created in the mRNA strand
With DNA polymerase I→ a nick translation
reaction synthesize the second cDNA strand

26. The mRNA is degraded by
Alkaline hydrolysis (NaOH)
• resulting in a single stranded
cDNA molecule that has a short
hairpin loop providing a 3’OH
terminus to be used by DNA
polymerase, klenow fragment or
reverse transcriptase
RNase H
• resulting in several nicks in the
mRNA strand to be used by DNA
polymerase I in a nick translation
reaction producing the 2nd
cDNA strand.

27. The mRNA is degraded by
Alkaline hydrolysis (NaOH) RNase H
The S1 nuclease is
used to digest the
polyA - polyT
tails and the hairpin
loop.

28. 4)cDNA cloning 2nd strand synthesis
mRNA may be
degraded by
Rnase H

29. 4)cDNA cloning 2nd strand synthesis
mRNA may be
degraded by
NaOH

30. • Single polypeptide chain resulted from the cleavage of DNA
polymerase I with subtilisin (large fragment of DNA polymerase I)
• The controlled use of proteases like subtilisin in molecular biology
allows researchers to modify enzymes to have specific properties,
facilitating their use in various experimental techniques and
applications.
Klenow fragment

31. • Enzymatic activity:
➢5' to 3' polymerase
➢3' to 5' exonuclease activity.
Lacks:
➢5’ to 3’
Klenow fragment
Researchers often choose the Klenow fragment
for specific experiments where the absence of
5'→3' exonuclease activity is advantageous:
• In the preparation of blunt-ended DNA
fragments for cloning
• In the synthesis of complementary DNA
(cDNA) during reverse transcription. The
controlled removal of specific enzymatic
activities allows scientists to tailor the
properties of the Klenow fragment to suit
their experimental needs

32. DNA POL I
subtilisin
Klenow
fragment

33. Uses
Labeling DNA by primer extension
(oligolabelling).
Filling sticky ends.
Proof-reading (correcting mis-matched
nucleotides)
cDNA cloning 2nd strand synthesis
Sequencing DNA (enzymatic dideoxy method –
Sanger and Coulson)

34. Labeling DNA by primer extension
(oligolabelling).

35. Labeling DNA by primer extension
(oligolabelling).

36. Labeling DNA by primer extension
(oligolabelling).

37. Sequenase
• Extracted from bacteriophage
T7.
• Enzymatic activity
➢Has high processivity and no
exonuclease activity.
➢5' to 3' polymerase activity and
fastest DNA polymerase
uses
❑Able to use modified
nucleotides as substrate

38. Taq polymerase
• Extracted from Thermus aquaticus.
• Thermo-stable DNA polymerase
(able to function at temp. higher
than 37°C).
• Optimum working temp. 72°C.
Uses
❑ Polymerase chain reaction
(PCR).

39. Enzymatic activity
It lacks a 3' to 5' exonuclease proofreading
activity
has an error rate measured at about 1 in 9,000
nucleotides
relatively low replication fidelity
Using other thermostable DNA
polymerases possessing a
proofreading activity
isolated from other thermophilic
bacteria and archaea,
instead of (or in combination
with) Taq
for high-fidelity amplification
Ex: Pfu DNA polymerase

40. Reverse transcriptase
• RNA dependent DNA polymerase
• Extracted from avian myeloblastosis
virus.
• Double polypeptide chain.
• Degrades RNA in a DNA-RNA hybrid.
• Enzymatic activity
➢ 5' to 3' polymerase, 5' to 3' and 3' to 5'
riboexonuclease activity.
Uses
❑cDNA cloning both strands
synthesis.
❑ Hybridization probes synthesis .
❑Labelling the termini of DNA
fragments with protruding 5'ends
(filling reaction ).