restriction enzymes,blotting technique

1. Restriction Enzymes
S.Manigandan
Ksr college of technology

2. Discovery
• Arbor and Dussoix in 1962 discovered that certain bacteria contain
Endonucleases which have the ability to cleave DNA.
• In 1970 Smith and colleagues purified and characterized the cleavage
site of a Restriction Enzyme.
• Werner Arbor, Hamilton Smith and Daniel Nathans shared the 1978
Nobel prize for Medicine and Physiology for their discovery of
Restriction Enzymes.

3. Biological Role
• Most bacteria use Restriction Enzymes as a defence
against bacteriophages.
• Restriction enzymes prevent the replication of the
phage by cleaving its DNA at specific sites.
• The host DNA is protected by Methylases which add
methyl groups to adenine or cytosine bases within the
recognition site thereby modifying the site and
protecting the DNA.

4. Restriction Enzymes
• Enzyme that cuts DNA at specific nucleotide sequences known as
restriction sites.
• Found in bacteria and have evolved to provide a defense mechanism
against invading viruses.
• In bacteria they selectively cut up foreign DNA in a process called
restriction
• To cut the DNA, restriction enzyme makes two incisions, each strand
of the DNA double helix.

5. Examples:
Enzyme Organism from which derived
Target sequence
(cut at *)
5' -->3'
Bam HI Bacillus amyloliquefaciens G* G A T C C
Eco RI Escherichia coli RY 13 G* A A T T C
Hind III Haemophilus inflenzae Rd A* A G C T T
Mbo I Moraxella bovis *G A T C
Pst I Providencia stuartii C T G C A * G
Sma I Serratia marcescens C C C * G G G
Taq I Thermophilus aquaticus T * C G A
Xma I Xanthamonas malvacearum C * C C G G G

6. • Restriction Enzymes scan the DNA sequence
• Find a very specific set of nucleotides
• Make a specific cut

7. Palindromes in DNA sequences
Genetic palindromes
are similar to verbal
palindromes. A
palindromic sequence
in DNA is one in which
the 5’ to 3’ base pair
sequence is identical
on both strands.
5
’
5
’
3’
3’

8. Restriction enzymes recognize and
make a cut within specific
palindromic sequences, known as
restriction sites, in the DNA. This is
usually a 4- or 6 base pair sequence.

9. Each of the double strands
of the DNA molecule is
complimentary to the
other; thus adenine pairs
with thymine, and
guanine with cytosine.

10. Restriction Endonuclease Types
Type I- multi-subunit, both endonuclease and methylase activities,
cleave at random up to 1000 bp from recognition sequence
Type II- most single subunit, cleave DNA within recognition sequence ,
cleave up to 4-8 bp from recognition sequence
Type III- multi-subunit, endonuclease and methylase about 25 bp from
recognition sequence

11. Once the recognition site was found
HaeIII could go to work cutting
(cleaving) the DNA
5’ TGACGGGTTCGAGGCCAG 3’
3’ ACTGCCCAAGGTCCGGTC 5’

12. These cuts produce what scientists call
“blunt ends”
5’ TGACGGGTTCGAGG CCAG 3’
3’ ACTGCCCAAGGTCC GGTC 5’

13. The names for restriction enzymes come from:
• the type of bacteria in which the enzyme is found
• the order in which the restriction enzyme was identified
and isolated.
EcoRI for example
R strain of E.coli bacteria
I as it is was the first E. coli restriction enzyme to
be discovered.

14. “blunt ends” and “sticky ends”
Remember how HaeIII produced a “blunt end”?
EcoRI, for instance, makes a staggered cut and
produces a “sticky end”
5’ GAATTC 3’
3’ CTTAAG 5’
5’ GAATTC 3’
3’ CTTAAG 5’
5’ G AATTC 3’
3’ CTTAA G 5’

15. blunt end
sticky end

16. Example of RE that produce sticky end cutters:

17.  DNA modifying enzymes
Alkaline phosphatase(notes)
DNA polymerase 1(notes)
Terminal transferase (notes)
S1 nuclease
Poly nucleotide kinase
Ribonuclease H
Ligase

18. Alkaline phosphatase

19. DNA polymerase 1
• Source is Ecoli
• Addition of nucleotide base pair of DNA
• It needa presence of primer
• It converts single stand DNA to double stand
• It has three subunits

20. Terminal transferase
(deoxy nucleotidyl transferase)
• It acts nucleotide to the 3! End to
the DNA molecules • 5! 3! 5! 3!
•
• 3! 5! 3! 5!
• 5! A(A)Na 5! T(T)nT
• A(A)Na T(T)Nt 3!
• 5! 3!
• 3! 5!

21. 4 >>S1 nuclease - Aspergillus oryzae.
 degrade single stranded DNA or RNA molecules.
 duplex of DNA and/or RNA strands has single stranded
overhangs or unhybridized internal loops - these will be
digested away.
 monomeric protein of a molecular weight of 38
kilodalton.
 requires Zn2+ as a cofactor and stable against denaturing
agents like urea, SDS, or formaldehyde.
 The optimum pH - 4-4.5.

23. 5>>POLYNUCLEOTIDE KINASE

24.  Transfer of the terminal phosphate of
ATPto
5' hydroxyl termini of polynucleotides
such as DNA and RNA, oligonucleotides,
and 3' mononucleotides.
 Polynucleotide kinase - T7 bacteriophage
(or T4 bacteriophage) enzyme that
catalyzes the transfer of a gamma-
phosphate from ATP to the free hydroxyl
end of the 5' DNA or RNA.
 This enzyme is commonly used to label
DNA or RNA with 32P or 33P at 5' ends.

26.  "forward reaction", PNK transfers the
gamma phosphate from ATP to the 5' end of a
polynucleotide (DNA or RNA).
 In the "exchange reaction", target DNA or
RNA that has a 5' phosphate is incubated with
an excess of ADP - in this setting, PNK will
first transfer the phosphate from the nucleic
acid onto an ADP, forming ATP and leaving a
dephosphorylated target. PNK will then
perform a forward reaction and transfer a
phosphate from ATP onto the target nucleic
acid.

27. 6>> Ribonuclease H
 (RNase H) is a family of non-sequence-
specific endonucleases that catalyze the
cleavage of RNA via a hydrolytic
mechanism.
 In DNA replication, RNase H is
responsible for removing the RNA
primer, allowing completion of the newly
synthesized DNA.

29. 7>> Ligase
 That facilitates the joining of DNA strands
together by catalyzing the formation of a
phosphodiester bond.
 DNA ligase is used in both DNA repair and DNA
replication .
 Purified DNA ligase is used in gene cloning to join
DNA molecules together to form recombinant
DNA.

30. Types of ligase:
 Prokaryates:
 The first uses NAD+ as a cofactor and only found in
bacteria.
 Eukaryates:
 The second uses ATP as a cofactor and found in
eukaryotes, viruses and bacteriophages. T4-
monomer with a molecular weight of 68 ± 6.8 kDa.
 The smallest known ATP-dependent DNA ligase is
the one from the bacteriophage T7 (at 41Kd).

31.  joining of DNA strands together by catalyzing the formation of
a phosphodiester bond between 3' hydroxyl ends of one
nucleotide, with the 5' phosphate end of another .
 ATP is required for the ligase reaction, which proceeds in
three steps:
 Adenylation (addition of AMP) of a lysine residue in the active
center of the enzyme, pyrophosphate is released;
 Transfer of the AMP to the 5' phosphate of the so-called
donor, formation of a pyrophosphate bond;
 Formation of a phosphodiester bond between the 5'
phosphate of the donor and the 3' hydroxyl of the acceptor.

32. THE MECHANISM OF DNA JOINING BY DNA LIGASE

33.  Temperature optimum for the ligation of nicked
DNA - 37 ◦C (at this temperature the hydrogen
bonding between the sticky ends is unstable).
 The optimum temperature for ligating the sticky
ends of restriction enzyme generated DNA
fragment - 4–20 ◦C.
 DNA ligase from E. coli will only ligate blunt-ended
DNA fragments - at a very high concentration.

 The ligase enzyme from bacteriophage T4 will
ligate blunt-ended DNA fragments, although at low
efficiency with respect to the efficiency of ligation of
sticky ended DNA fragments.

34. BASICS OF CLONING

35. Breaking and joining DNA using restriction enzymes and DNA ligase. Linear DNA (insert) and a
closed-circular plasmid DNA (vector) each contain the recognition site for BamHI and EcoRI.
Mixing the DNA fragments with compatible ends together in the presence of DNA ligase can
result in the formation of vector–insert hybrid DNA molecules

36. The basics of cloning into a plasmid vector containing a single unique restriction enzyme recognition site. The vector
contains a single EcoRI recognition site, while the insert has two. Cutting both with the enzyme generates the fragments
shown. Adding ligase to the cut vector will probably result in its reformation. This can be prevented by treating the cut
vector with phosphatase to remove the 5 phosphate residues from the ends of the DNA. The cut insert can provide the
missing phosphate that DNA ligase requires, so mixing the vector and insert will result in the formation of hybrid DNA
molecules
Vector with insert Reformed vector

37. ADAPTORS
Adaptors, like linkers, are short synthetic
oligonucleotides.
But unlike linkers, an adaptor is synthesized so that
it already has one sticky end

39. LINKERS-
short pieces of double stranded
DNA, of known nucleotide
sequence, that are synthesized in
the test tube.

41. MODIFICATION SYSTEMS
DNA adenine methyltransferase
DNA cytosine
methyltransferase
Modified cytosine restriction

42. Methylase - an enzyme that adds a
methyl group to a molecule; in
restriction-modification systems of
bacteria a methyl group is added to
DNA at a specific site to protect the
site from restriction endonuclease
cleavage .

43. DNA methyltransferases - transfer a
methyl group from S-adenosylmethionine
to either adenine or cytosine residues.
Self protection of its own DNA from the
attack of restriction enzymes.
Most laboratory strains of E. coli contain
three site-specific DNA methylases.

44.  The dam methylase of E. coli recognizes the
tetranucleotide GATC in DNA and transfers a
methyl group (from S-adenosyl methionine) to
the amino group at position 6 of the adenine in
that sequence.
 Dcm methyltransferases–methylation at
the C5 position of the second cytosine in
the sequences CCAGG and CCTGG.
 EcoKI methylase–methylation of adenine
in the sequences AAC(N6)GTGC and
GCAC(N6)GTT.

45. DAM - Deoxyadenosine methylase -
enzyme that adds a methyl group to
the adenine of the sequence 5'-
GATC-3 ‘in newly synthesized DNA.
Immediately after DNA synthesis, the
daughter strand remains
unmethylated for a short time.

47. DAM METHYLATES ADENINE OF GATC SITES AFTER REPLICATION

49. Mcr system - modified
cytosine
restriction

50. DNA from various bacterial and eukaryotic sources
could only be cloned at very low efficiency in certain E.
coli strains, the problem being that the incoming DNA
was being restricted by the host. The phenomenon is
caused by methylcytosine in DNA, and is called
modified cytosine restriction (Mcr).
One Mcr system is encoded by two genes, McrB and
McrC, from within the E. coli genome, close to the site
of the EcoKI restriction–modification system .The
proteins encoded by these genes recognize the DNA
sequence 5-R–mC–N40–80–R–mC-3, where R is either
A or G,N is any nucleotide and mC is methylated
cytosine. Cleavage occurs at multiple sites in both
strands between the methylated cytosine residues,and
requires GTP to translocate DNA between the two 5-
RmC-3 binding sites.

51. The Dam- and Dcm-dependent methylation
may interfere with cleavage by restriction
endonucleases .
For example, MboI (recognition sequence 5-
GATC-3) does not cut DNA methylated by the
Dam methylase, while its isoschizomer
Bsp143I is insensitive to Dam methylation.
Similarly, EcoRII (5CCWGG-3- where w= A or
T) does not cleave Dcm methylated DNA,
meanwhile its isoschizomer MvaI does.

52. Restriction – modification systems found in laboratory E. coli K strains.The genomic location of
restriction – modification systems in E. coli K12. The circle represents the genome of E. coli, with
the numbers on the inside indicating genomic location. E. coli contains two type II modification
systems (Dam and Dcm) and two type I restriction – modification systems (Mcr and EcoKI). The
various methylase, restriction and specificity subunits are colour coded as indicated

53. BLOTTING TECHNIQUES

54. What is blotting?
Blots are techniques for transferring DNA , RNA and
proteins onto a carrier so they can be separated, and
often follows the use of a gel electrophoresis.
 The Southern blot is used for transferring DNA, the
Northern blot for RNA and the western blot for
PROTEIN.

55. TYPES OF BLOTTING TECHNIQUES
Blotting technique
Southern Blot
It is used to detect DNA.
Northern Blot
It is used to detect RNA.
Western blot
It is used to detect protein.

56. SOUTHERN BLOTTING
• This method Involves separation, transfer and hybridization.
• It is a method routinely used in molecular biology for detection
of a specific DNA sequence in DNA samples. The DNA detected
can be a single gene, or it can be part of a larger piece of DNA
such as a viral genome.

57. CONT………..
• Southern blotting combines agarose gel electrophoresis for size separation of
DNA with methods to transfer the size separated DNA to a filter membrane
for probe hybridization.
• The key to this method is Hybridization.
• Hybridization - Process of forming a double-stranded DNA molecule between a
single-stranded DNA probe and a single-stranded target patient DNA.

58. PRINCIPLE
1. The mixture of molecules is separated.
2. The molecules are immobilized on a matrix.
3. The probe is added to the matrix to bind to the
molecules.
4. Any unbound probes are then removed.
5. The place where the probe is connected corresponds to
the location of the immobilized target molecule.

59. Capillary plotting apparatus

60. Steps in southern blotting
1. Digest the DNA with an
appropriate restriction
enzyme.
2.The complex mixture of
fragments is subjected to gel
electrophoresis to separate
the fragments according to
size.

61. Cont….
3.The restriction fragments
present in the gel are
denatured with alkali and
transferred onto
4. a nitrocellulose filter or nylon
membrane by blotting.
• This procedure preserves the
distribution of the fragments in
the gel, creating a replica of the
gel on the filter.

62. Cont….
5.The filter is incubated under
hybridization conditions with
a specific radiolabeled DNA
probe.
• The probe hybridizes to the
complementary DNA
restriction fragment.

63. Cont….
6.Excess probe is washed away and the
probe bound to the filter is detected by
autoradiography, which reveals the DNA
fragment to which the probe
hybridized.

66. APPLICATIONS
• Southern blots are used in gene discovery , mapping, evolution and
development studies, diagnostics and forensics (It is used for DNA
fingerprinting, preparation of RFLP maps)
• identification of the transferred genes in transgenic individuals, etc.

67. APPLICATIONS
• Southern blots allow investigators to determine the molecular weight of a
restriction fragment and to measure relative amounts in different samples.
• Southern blot is used to detect the presence of a particular bit of DNA in a
sample
• analyze the genetic patterns which appear in a person's DNA.

68. Northern Blotting
Northern blotting is a technique for detection of
specific RNA sequences. Northern blotting was
developed by James Alwine and George Stark at
Stanford University (1979) and was named such
by analogy to Southern blotting

69. Steps involved in Northern blotting
1. RNA is isolated from several
biological samples (e.g. various
tissues, various developmental
stages of same tissue etc.)
* RNA is more susceptible to
degradation than DNA.

70. Cont……
2. Sample’s are loaded on gel and
the RNA samples are separated
according to their size on an
agarose gel .
• The resulting gel following after
the electrophoresis run.

71. Cont……
3. The gel is then blotted
on a nylon membrane or
a nitrocellulose filter
paper by creating the
sandwich arrangement.

72. Cont……
4. The membrane is placed in a dish
containing hybridization buffer with a
labeled probe.
• Thus, it will hybridize to the RNA on the
blot that corresponds to the sequence of
interest.
5. The membrane is washed to remove
unbound probe.

73. Cont……
6. The labeled probe is detected via
autoradiography or via a
chemiluminescence reaction (if a
chemically labeled probe is used). In both
cases this results in the formation of a
dark band on an X-ray film.
• Now the expression patterns of the
sequence of interest in the different
samples can be compared.

74. APPLICATIONS
• A standard for the study of gene expression at the level of mRNA (messenger RNA transcripts)
• Detection of mRNA transcript size
• Study RNA degradation
• Study RNA splicing
• Study RNA half-life
• Often used to confirm and check transgenic / knockout mice (animals)

75. Disadvantage of Nourthern
plotting
1.The standard northern blot method is relatively less sensitive than nuclease
protection assays and RT-PCR
2. Detection with multiple probes is a problem
3. If RNA samples are even slightly degraded by RNases, the quality of the data and
quantitation of expression is quite negatively affected.

76. Western blotting
• Western blotting (1981) is an Immunoblotting technique
which rely on the specificity of binding between a protein of
interest and a probe (antibody raised against that particular
protein) to allow detection of the protein of interest in a
mixture of many other similar molecules.
• The SDS PAGE technique is a prerequisite for Western
blotting .

77. Steps in western blotting
1. A protein sample is subjected to
electrophoresis on an SDS-
polyacrylamide gel.
2. Electroblotting transfers the
separated proteins from the gel to
the surface of a nitrocellulose
membrane.

78. Cont…
3. The blot is incubated with a generic protein (such as milk
proteins or BSA) which binds to any remaining sticky
places on the nitrocellulose.
4. An antibody that is specific for the protein of interest (the
primary antibody - Ab1) is added to the nitrocellulose
sheet and reacts with the antigen. Only the band
containing the protein of interest binds the antibody,
forming a layer of antibody molecules .

79. Cont…
5. After washing for removal of non-specifically
bound Ab1, second antibody (Ab2)is added,
which specifically recognizes the Fc domain of
the primary antibody and binds it. Ab2 is
radioactively labeled, or is covalently linked to a
reporter enzyme, which allows to visualize the
protein-Ab1-Ab2 complex.

80. Application
1.The confirmatory HIV test
2.Western blot is also used as the definitive test for Bovine spongiform encephalopathy (BSE(
3.Some forms of Lyme disease testing employ Western blotting .