This document discusses various enzymes used for gene cloning. It describes restriction endonucleases that cut DNA at specific recognition sequences, leaving sticky or blunt ends. DNA ligase joins compatible DNA ends. DNA polymerases synthesize new DNA strands. Other enzymes mentioned include DNA modifying enzymes, topoisomerases, and enzymes for tailing DNA ends. The document provides examples of various restriction enzymes and their recognition sequences. It explains how enzymes are used in gene cloning techniques.

1. SUBMITTED TO- Proff. P.P.Balgir
SUBMITTED BY-NEHA SINGLA
MSc. ( Hon’s) Biotechnology
ROLL NO -18011001

2. • Enzymes for gene cloning
-Nucleases
-restriction endonucleases
-DNA ligase
-DNA polymerase
-polynucleotide kinases
-Terminal Deoxynucleotidyl Transferase
-Modification of blunt ends
- Topoisomerase

3. Enzymes for Gene Cloning

4. Gene Cloning

5. • To break phosphodiester bond.
Two types-
1.EXONUCLEASES
2.ENDONUCLEASES
• Exonuclease degrade nucleic acids from one end of the
molecule. They operate either in 5’to3’ or 3’ to5’
direction. For example-snake venom
phosphodiesterase and bovine spleen
phosphodiesterase
• Endonucleases degrade nucleic acids at specific
internal sites, reducing it to smaller and smaller
fragments.
Restriction enzymes, an endonucleases are important in

6. • Aspergillus nuclease S1 is used in the laboratory as a
reagent in nuclease protection assays. In molecular
biology, it is used in removing single stranded tails from
DNA molecules to create blunt ended molecules and
opening hairpin loops generated during synthesis of
double stranded cDNA.

7. • Bacteria have learned to "restrict" the possibility of attack
from foreign DNA by means of "restriction enzymes”.
• Cut up “foreign” DNA that invades the cell.
• Type II restriction enzymes cleave DNA chains at selected
sites.
• Enzymes may recognize 4, 6 or more bases in selecting
sites for cleavage.

10. Discovery of restriction enzymes and their application to problems of molecular
genetics
Biozentrum der
Universität, Basel,
Switzerland
Johns Hopkins
University School of
Medicine, Baltimore,
MD, USA
Johns Hopkins
University School of
Medicine, Baltimore,
MD, USA

12. One or two genes encoding protein that cleave only
modified DNA , including methylated , hydroxymethylated
and glucosyl hydroxymethylated bases.

13. • Restriction endonucleases appear to function by
‘scanning’ the length of a DNA molecule by binding to it in
a non-specific.
• Once it encounters its particular recognition sequence,
the restriction enzyme undergoes a large conformational
change, which activates the catalytic sites.
• The enzyme will then make one cut in each of the two
sugar–phosphate backbones of the DNA double helix to
generate a 3’ hydroxyl and a 5’ phosphate

14. • No ATP requirement.
• Recognition sites in double stranded DNA have a 2-fold
axis of symmetry – a “palindrome”.
• Cleavage can leave staggered or "sticky" ends or can
produce "blunt” ends.

15. Cuts usually occurs at
a palindromic sequence
SmaI: produces blunt ends
5´ CCCGGG 3´
3´ GGGCCC 5´
EcoRI: produces sticky ends
5´ GAATTC 3´
3´ CTTAAG 5´
Examples of Palindromes:
Don't nod
Dogma: I am God
Never odd or even

16. • EcoRI – Escherichia coli strain RY13, 1st enzyme
• BamHI – Bacillus amyloliquefaciens strain H, 1st enzyme
• HindIII – Haemophilus influenzae, strain Rd, 3rd enzyme
• Sau3AI – Staphylococcus aureus strain 3A, 1st enzyme

19. EcoR l, one of the earliest restriction enzymes
identified was isolated from E. coli.
5’ GAATTC 3’
3’ CTTAAG 5’
The recognition sequence for EcoRl reads the
same in the 5' - 3' direction on one strand as it
does in the 5' - 3' direction on the other strand
– Palindrome sequence.

20. STICKY END-
The DNA fragments produced by EcoRl
digestion have overhanging single-stranded
tails ("sticky ends") that can anneal with
a complementary over hanging tails on other
DNA fragments.

21. BLUNT END-
Restriction enzymes (Sma l), cleave
DNA to produce blunt-ended fragments.

24. • Enzymes that cut staggered cuts result in
complementary ends that can be ligated together.
• HindIII
5’ --A AGCTT--3’
5’ --AAGCTT-- 3’
3’ --TTCGA A--5’
3’ --TTCGAA-- 5’
• Sticky ends that are complementary (from digests with
the same or different enzymes) can be ligated together.
• Sticky ends that are not complementary cannot be
ligated together.

25. DNA Ligase can also join blunt ends

26. • The DNA ligase from bacteriophage T4 is the ligase most-
commonly used in laboratory research.
• It can ligate cohesive or "sticky" ends of DNA,
oligonucleotides, as well as RNA and RNA-DNA hybrids,
but not single-stranded nucleic acids.
• It can also ligate blunt-ended DNA with much greater
efficiency than E. coli DNA ligase. Unlike E. coli DNA
ligase, T4 DNA ligase cannot utilize NAD and it has an
absolute requirement for ATP as a cofactor.

27. • BamHI -G
GATCC-
-CCTAG
G-
• BglII -A
GATCT-
-TCTAG
A-
• Result -GGATCT-
-CCTAGA-
No longer palindromic,
so not cut by BamHI or
BglII

31. DNA POLYMERASES

32. 1. DNA dependent DNA Polymerases
2. RNA dependent DNA Polymerases
Primer, dNTPs and cofactors are required

33. • 5’ to 3’ Polymerase activity
• 5’ to 3’ exonuclease activity
• 3’ to 5’ exonuclease activity

34. 1.DNA Polymerase (E.coli)
( Klenow fragment-76KDa piece)
2. Taq Polymerase (for synthesis of longer stretches of
DNA)
3. T4 DNA Polymerase ( More active 3’ to 5’
exonuclease activity, for polishing sticky ends)
4. T7 DNA Polymerase ( No 5’ to 3’ exonuclease
activity, used in DNA sequencing)

35. • Also known as reverse transcriptases
• Template is RNA instead of DNA
• Example- RTases from AMV-Alfalafa mosaic virus(pol gene
product)
• Uses- cDNA library construction, RNA sequencing
• No proofreading (3’ to 5’ exonuclease activity) so error rate high
• (1 in 500).

37. • The T4 Polynucleotide Kinase (T4 PNK) catalyzes the
transfer of the phosphate from ATP to the 5'-OH group of
single- and double-stranded DNAs and RNAs as well as
oligonucleotides. The reaction can be reversed.
• The enzyme is also a 3'-phosphatase and a homotetramer. It
consists of four identical subunits of 28.9 kDa.
• It can be found in E. coli with a duplicated gene of the
bacteriophage T4.
• It can also be used for labeling 5’-terminus of nucleic acids
which can be used as probes for hybridization, transcript
mapping; markers for gel electrophoresis.
• They can also be used as primers for DNA sequencing and
PCR technique as well as an enzyme that removes the 3’-
phosphate groups.

38. • In enzymology, a polynucleotide 5'-hydroxyl-kinase (EC
2.7.1.78) is an enzyme that catalyzes the chemical reaction
ATP + 5'-dephospho-DNA  ADP + 5'-phospho-DNA

40. • Terminal Deoxynucleotidyl Transferase (TdT), a template-
independent DNA polymerase, catalyzes the repetitive
addition of deoxyribonucleotides to the 3'-OH of
oligodeoxyribonucleotides and single-stranded and double-
stranded DNA
• Use- Homopolymeric tailing of linear duplex DNA with any
type of 3'-OH terminus
• TdT is unique in its ability to use a variety of divalent
cations such as Co2+, Mn2+, Zn2+ and Mg2+

43. • E.coli cells carrying a cloned gene encoding calf thymus
terminal deoxynucleotidyl transferase
• Terminal Deoxynucleotidyl Transferase is purified from a
baculovirus clone of calf thymus TdT.
• It has a molecular weight of 58 kDa.

45. Modification of Blunt ends

46. LINKERS

47. Possible problem with use of
LINKERS

48. ADAPTORS

51. HOMOPOLYMER TAILING

53. Alkaline phasphatases
Removes phosphate groups from 5' ends of DNA
(prevents unwanted re-ligation of cut DNA)
DNA Ligase
Joins compatible ends of DNA fragments
(blunt/blunt or complementary cohesive ends).
Uses ATP
DNA Polymerase
Synthesises DNA complementary to a DNA
template in the 5'-to-3'direction. Starts from an
oligonucleotide primer with a 3' OH end
Exonuclease III
Digests nucleotides progressively from a DNA
strand in the 3' -to-5' direction
Polynucleotide Kinase
Adds a phosphate group to the 5' end of double- or
single-stranded DNA or RNA. Uses ATP
RNase A Nuclease which digests RNA, not DNA
Taq DNA Polymerase
Heat-stable DNA polymerase isolated from a
thermostable microbe (Thermus aquaticus)

54. • Topoisomerase are enzymes that regulate the
overwinding or underwinding of DNA
• Topoisomerases are isomerase enzymes that act on the
topology of DNA.
ROLE OF TOPOISOMERASE-
• The winding problem of DNA arises due to the interwined
nature of its double helical structure.
• During DNA replication and transcription , DNA becomes
overbound ahead of replication fork.
• If left unabated , this torsion would eventually stop the
ability of RNA and DNA polymerase involved in these
processes to continue down the DNA strand.

55. • Topoisomerase bind to either single stranded or double
stranded DNA and cut the phosphate backbone of the
DNA.
• This intermediate break allows the DNA to be untangled
or unwound , and, at the end of these processes, the
DNA backbone is resealed again.
Types of topoisomerases-
• Topoisomerase I- BREAKS ONLY ONE STRAND
• Topoisomerase II – BREAKS BOTH STRANDS.

56. • Type I topoisomerases are enzymes that cut one of the
two strands of double stranded DNA , relax the strand ,
and reanneal.
Type II topoisomerases-
Type II topoisomerases cut both strands of the DNA helix
simultaneously. They use the hydrolysis of ATP, unlike type
I topoisomerase. These enzymes change the linking
number of circular DNA by 2

58. • To remove DNA supercoils during transcription and DNA
replication
• For strand breakage during recombination.
• For chromosomal condensation and to distangle
interwined DNA during mitosis

59. • GENE CLONING – T.A. Brown
• Molecular Biotechnology: Principles and Applications of
Recombinant DNA by B.R. Glick and J.J. Pasternak,
ASM Press, USA (2010).

60. Thank you