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DNA SEQUENCING, MODIFICATION & RESTRICTION




                                             2
3
 DNA sequencing is the determination of the order of bases in
sample of DNA.

 It is the reading of the genetic code.

 However, not all DNA sequences are genes (i.e. coding regions)
as there may, depending on the organism and the source of the
DNA sample, also be promoters, tandem repeats, introns, etc.




                                                               4
Two methods for the large-scale sequencing of DNA became
available in the late 1970's.

Both based on generation of DNA fragments of different
lengths which start at a fixed point and terminate at specific
nucleotides.

DNA fragments are separated by size on polyacrylamide
gels and the nucleotide sequences are directly read from the
gel.



                                                                 5
1. Maxam-Gilbert sequencing (chemical cleavage method
using double-stranded (ds) DNA) in which the sequence of a
double-stranded DNA molecule is determined by treatment
with chemicals that cut the molecule at specific nucleotide
positions.




                                                              6
2.  Sanger-Coulson sequencing (chain termination method
using single-stranded (ss) DNA) in which the sequence of a
single-stranded DNA molecule is determined by enzymatic
synthesis of complementary polynucleotide chains, these chains
terminating at specific nucleotide positions.




                                                           7
8
Sanger Method of DNA
               Sequencing
Major steps

1.   Template DNA (ssDNA)
2.   Primer annealing
3.   Complementary strand synthesis
4.   Labeling for the detection of fragments
5.   Chain termination using ddNTPs
6.   Resolution on denaturing PAGE
7.   Visualization of bands by autoradiography




                                                 9
 preparation of identical single-stranded DNA molecules.

The first step is to anneal a short oligonucleotide to the same
position on each molecule, this oligonucleotide subsequently acting
as the primer for synthesis of a new DNA strand that is
complementary to the template which is to be sequenced .

The strand synthesis reaction catalyzed by a DNA polymerase
enzyme and requires the four deoxyribonucleotide triphosphates
(dNTPs - dATP, dCTP, dGTP and dTTP) as substrates, would
normally continue until several thousand nucleotides had been
polymerized.




                                                               10
This does not occur in a chain termination sequencing
experiment because, as well as the four dNTPs, a small amount
of a dideoxynucleotide (e.g. ddATP) is added to the reaction.

 The polymerase enzyme does     not discriminate between
dNTPs and ddNTPs, so the dideoxynucleotide can be
incorporated into the growing chain, but it then blocks further
elongation because it lacks the 3′-hydroxyl group needed to
form a connection with the next nucleotide




                                                                  11
12
13
14
Reading a DNA Sequencing Gel




                       Sequence 5’ to 3’
                   C
                   G
                   G
                   G
                   C
                   G
                   T

                                           15
The smallest fragments will be at the bottom of the gel, the
largest fragments at the top.
The DNA sequence can be determined by determining the
terminating base for the shortest fragment, then for the next
shortest fragment for all of the DNA fragments




                                                                16
MAXAM-GILBERT SEQUENCING

This chemical cleavage method uses double-stranded DNA
samples and so does not require cloning of DNA into an M13
phage vector to produce single-stranded DNA.

 It involves modification of the bases in DNA followed by
chemical base-specific cleavage.

Stages:

1. Double-stranded DNA to be sequenced is labeled by
attaching a radioactive phosphorus (32P) group to the 5' end.
Polynucleotide kinase enzyme and 32P-dATP is used here.

                                                             17
2. Using dimethyl sulphoxide (DMSO) and heating to
90oC, the two strands of the DNA are separated and purified
(e.g. using gel electrophoresis and the principle that one of
the strands is likely to be heavier than the other due to the
fact that it contains more purine nucleotides (A and G) than
pyrimidines (C and T) which are lighter.

3. Single-stranded sample is split into separate samples
and each is treated with one of the cleavage reagents. This
part of the process involves alteration of bases (e.g.
dimethylsulphate methylates guanine) followed by removal
of altered bases. Lastly, piperidine is used for cleavage of
the strand at the points where bases are missing
                                                         18
Chemical      Chemical
  Base      Chemical used for     used for     used for
specificity  base alteration    altered base    strand
                                  removal      cleavage



    G       Dimethylsulphate     Piperidine    Piperidine


  A+G             Acid             Acid        Piperidine

  C+T          Hydrazine         Piperidine    Piperidine

            Hydrazine + High
    C                            Piperidine    Piperidine
                  salt

  A>C            Alkali          Piperidine    Piperidine
                                                        19
G   A+G C +T   C   Sequence
                      C
                      G
                       T
                       T
                      C
                      C

                       G
                       G
                       A
                       C
                       T
                       A
                       A


                              20
Automated DNA Sequencing with
     Fluorescent Dyes




Each different ddNTP is coupled to a different colored
fluorescent dye
        ddTTP is red; ddGTP is black etc.                21
Alternative Sequencing Methods:
   Pyrosequencing


Pyrosequencing is based on the generation of light signal
through release of pyrophosphate (PPi) on nucleotide
addition.
   DNAn + dNTP  DNAn+1 + PPI
PPi is used to generate ATP from adenosine phosphosulfate
(APS).
   APS + PPI  ATP
ATP and luciferase generate light by conversion of luciferin to
oxyluciferin.


                                                              22
   Each nucleotide is added in turn.
   Only one of four will generate a light signal.
   The remaining nucleotides are removed enzymatically.
   The light signal is recorded on a pyrogram.



               DNA sequence: A T C A GG CC T




              Nucleotide added : A T C A G   C T

                                                           23
Bisulfite Sequencing
 Bisulfite sequencing is used to detect methylation in
  DNA.
 Bisulfite deaminates cytosine, making uracil.
 Methylated cytosine is not changed by bisulfite
  treatment.
 The bisulfite-treated template is then sequenced.




                                                          24
Bisulfite Sequencing
 The sequence of treated and untreated templates is
  compared.
                               Me         Me              Me
 Methylated sequence:    GTC        GGC        GATCTATC        GTGCA …
                               Me         Me              Me
 Treated sequence:       GTC        GGC        GATUTATC        GTGUA …

 DNA Sequence:

 (Untreated) reference: ...GTCGGCGATCTATCGTGCA…
 Treated sequence:      ...GTCGGCGATUTATCGTGUA…

      This sequence indicates that these Cs are methylated.
                                                                         25
Genome sequencing strategies

 Only short DNA molecules (~800 bp) can be sequenced
  in one read, so large DNA molecules, such as
  genomes, longer sequences must be subdivided into
  smaller fragments and subsequently reassembled to give
  the overall sequence.
 Genome sequencing can be approached in two ways




                                                        26
Whole-genome shotgun sequencing
The whole-genome shotgun approach was first
proposed by Craig Venter and colleagues as a
means of speeding up the acquisition of
contiguous sequence data for large genomes
such as the human genome and those of other
eukaryotes (Venter et al., 1998; Marshall




                                               27
Clone contig sequencing:
 Involves the systematic production and
  sequencing of sub clones arrange overlapping
  clones before sequencing.




                                                 28
DNA MODIFICATION & RESTRICTION

 Bacteria can destroy an invading or foreign DNA from an other
species, thus preventing its replication, transcription, or
incorporation in to the host cell genome.

This is made possible by an ingenious combination of two
enzymatic processes called modification & restriction.




                                                            29
MODIFICATION




               30
MODIFICATION
 It is the enzymatic alteration of its own DNA by the
  cell, in a species distinctive way , thus differentiating it
  from that of other species.
 The protective modification of the host cell DNA is
  brought about by modification methylases,which
  methylate certain adenine residues.
 Once the host cell DNA is modified in this manner,it
  cannot be degraded by that cells restriction enzymes.



                                                                 31
 The restriction methylases transfer methyl groups
  from s-adenosylmethionine to pairs of adenine
  residues in duplex DNA , one in each strand; the two
  adenine are on adjacent or near by base pairs.
 The sequence of bases on the two stands between and
  near the methylated adenines is symmetrical on either
  side of a mid point.




                                                          32
DNA RESTRICTION




                  33
A Restriction Enzyme (or restriction endonuclease) is an
enzyme that cuts double-stranded DNA at specific
recognition nucleotide sequences known as
restrictionsites.

 Inside a bacterial host, the restriction enzymes selectively
cut up foreign DNA in a process called restrication.

 To cut the DNA, a restriction enzyme makes two
incisions, once through each sugar-phosphate backbone
(i.e. each strand) of the DNA double helix.

                                                            34
35
Restriction Enzymes scan the
DNA sequence.




                               36
 recognition site
 5'-GTATAC-3'
 ::::::
 3'-CATATG-5'

 A palindromic recognition site reads the same on the
  reverse strand as it does on the forward strand when both
  are read in the same orientation.

 Restriction enzymes recognize a specific sequence of
  nucleotides and produce a double-stranded cut in theDNA.

 There are two types of palindromic sequences that can be
  possible in DNA.

                                                              37
The Mirror like palindrome is similar to those found in
 ordinary text, in which a sequence reads the same forward
 and backwards on a single strand of DNA strand, as in
 GTAATG.

 The inverted repeat palindrome is also a sequence that
 reads the same forward and backwards, but the forward
 and backward sequences are found in complementary
 DNA strands (i.e., of double-stranded DNA), as in GTATAC
 (GTATAC being complementary to CATATG).

 Inverted repeat palindromes are more common and have
 greater      biological   importance   than   mirror-like
 palindromes.

                                                         38
 Different restriction enzymes that recognize the same
  sequence are known as neoschizomers.

 These often cleave in different locales of the sequence.
  Different enzymes that recognize and cleave in the same
  location are known as isoschizomers.

 Types
 Restriction endonucleases are categorized into three or
  four general groups (Types I, II and III) based on their
  composition and enzyme cofactor requirements, the nature
  of their target sequence, and the position of their DNA
  cleavage site relative to the target sequence.


                                                             39
 There are four classes of restriction endonucleases:
 types I, II,III and IV. All types of enzymes recognise
 specific short DNA sequences and carry out the
 endonucleolytic cleavage of DNA to give specific
 double-stranded fragments with terminal 5'-
 phosphates

 They differ in their recognition sequence, subunit
 composition, cleavage position, and cofactor
 requirements


                                                          40
 Type I restriction enzymes were the first to be identified
  and were first identified in two different strains (K-12 and
  B) of E. coli.
 These enzymes cut at a site that differs, and is a random
  distance (at least 1000 bp) away, from their recognition
  site.
 Cleavage at these random sites follows a process of DNA
  translocation, which shows that these enzymes are also
  molecular motors.
 The recognition site is asymmetrical and is composed of
  two specific portions—one containing 3–4
  nucleotides, and another containing 4–5 nucleotides—
  separated by a non-specific spacer of about 6–8
  nucleotides.                                                 41
 These enzymes are multifunctional and are capable of
 both restriction and modification activities, depending
 upon the methylation status of the target DNA.

 The cofactors S-Adenosyl methionine (AdoMet),
 hydrolyzed adenosine triphosphate (ATP), and
 magnesium (Mg2+) ions, are required for their full
 activity.

 Type I restriction enzymes possess three subunits
 called HsdR, HsdM, and HsdS;

                                                           42
 HsdR is required for restriction; HsdM is necessary for
 adding methyl groups to host DNA (methyltransferase
 activity) and HsdS is important for specificity of the
 recognition (DNA-binding) site in addition to both
 restriction (DNA cleavage) and modification (DNA
 methyltransferase) activity.[




                                                            43
Type II:




           44
 They are a dimer of only one type of subunit; their
 recognition sites are usually undivided and
 palindromic and 4–8 nucleotides in length, they
 recognize and cleave DNA at the same site, and they
 do not use ATP or AdoMet for their activity—they
 usually require only Mg2+ as a cofactor.[


 These are the most commonly available and used
 restriction enzymes.



                                                        45
 In the 1990s and early 2000s, new enzymes from this
 family were discovered that did not follow all the
 classical criteria of this enzyme class, and new
 subfamily nomenclature was developed to divide this
 large family into subcategories based on deviations
 from typical characteristics of type II enzymes.




                                                        46
 Type IIB restriction enzymes (e.g. BcgI and BplI) are
  multimers, containing more than one subunit
 They cleave DNA on both sides of their recognition to cut
  out the recognition site.
 They require both AdoMet and Mg2+ cofactors. Type IIE
  restriction endonucleases (e.g. NaeI) cleave DNA
  following interaction with two copies of their recognition
  sequence.[
 One recognition site acts as the target for cleavage, while
  the other acts as an allosteric effector that speeds up or
  improves the efficiency of enzyme cleavage.


                                                           47
 Type IIG restriction endonucleases (Eco57I) do have a
  single subunit, like classical Type II restriction
  enzymes, but require the cofactor AdoMet to be active.

 Type IIM restriction endonucleases, such as DpnI, are able
  to recognize and cut methylated DNA

 Type IIS restriction endonucleases (e.g. FokI) cleave DNA
  at a defined distance from their non-palindromic
  asymmetric recognition sites

 These enzymes may function as dimers. Similarly, Type IIT
  restriction enzymes (e.g., Bpu10I and BslI) are composed of
  two different subunits

                                                               48
 Type III restriction enzymes (e.g. EcoP15) recognize two
    separate non-palindromic sequences that are inversely
    oriented. They cut DNA about 20-30 base pairs after the
    recognition site.

 These enzymes contain more than one subunit and require
    AdoMet and ATP cofactors for their roles in DNA
    methylation and restriction, respectively.

 They are components of prokaryotic DNA restriction-
    modification mechanisms that protect the organism
    against invading foreign DNA.


                                                              49
 Type III enzymes are hetero-
  oligomeric, multifunctional proteins composed of two
  subunits, Res and Mod.

 The Mod subunit recognises the DNA sequence
  specific for the system and is a modification
  methyltransferase; as such it is functionally equivalent
  to the M and S subunits of type I restriction
  endonuclease.

 Res is required for restriction, although it has no
  enzymatic activity on its own.

                                                             50
 Type III enzymes recognise short 5-6 bp long
  asymmetric DNA sequences and cleave 25-27 bp
  downstream to leave short, single-stranded 5'
  protrusions
 They require the presence of two inversely oriented
  unmethylated recognition sites for restriction to occur.
  These enzymes methylate only one strand of the
  DNA, at the N-6 position of adenosyl residues, so
  newly replicated DNA will have only one strand
  methylated, which is sufficient to protect against
  restriction.



                                                             51
 Type III enzymes belong to the beta-subfamily of N6
 adenine methyltransferases, containing the nine
 motifs that characterize this family, including motif
 I, the AdoMet binding pocket (FXGXG), and motif
 IV, the catalytic region (S/D/N (PP) Y/F).[




                                                         52
5'GGTACC   5'---GGTAC C---3'
               Klebsiella pneumoniae
                                       3'CCATGG   3'---C CATGG---5'

                                       5'CTGCAG   5'---CTGCA G---3'
PstI[48]       Providencia stuartii
                                       3'GACGTC   3'---G ACGTC---5'
               Streptomyces            5'GAGCTC   5'---GAGCT C---3'
SacI[48]
               achromogenes            3'CTCGAG   3'---C TCGAG---5'
                                       5'GTCGAC   5'---G TCGAC---3'
SalI[48]       Streptomyces albus
                                       3'CAGCTG   3'---CAGCT G---5'
               Streptomyces            5'AGTACT   5'---AGT ACT---3'
ScaI[48]
               caespitosus             3'TCATGA   3'---TCA TGA---5'
                                       5'ACTAGT   5'---A CTAGT---3'
SpeI           Sphaerotilus natans
                                       3'TGATCA   3'---TGATC A---5'
               Streptomyces            5'GCATGC   5'---GCATG C---3'
SphI[48]
               phaeochromogenes        3'CGTACG   3'---C GTACG---5'
               Streptomyces            5'AGGCCT   5'---AGG CCT---3'
StuI[49][50]
               tubercidicus            3'TCCGGA   3'---TCC GGA---5'
                                       5'TCTAGA   5'---T CTAGA---3'
                                                                 53
XbaI[48]       Xanthomonas badrii
Enzyme              Source              Recognition Sequence              Cut

                                                                        5'---G     AATTC---
                                               5'GAATTC                 3'
EcoRI             Escherichia coli
                                               3'CTTAAG                 3'---CTTAA     G---
                                                                        5'

                                               5'CCWGG                  5'---     CCWGG---3'
EcoRII            Escherichia coli
                                               3'GGWCC                  3'---GGWCC     ---5'

                                                                        5'---G     GATCC---
                                               5'GGATCC                 3'
BamHI             Bacillus amyloliquefaciens
                                               3'CCTAGG                 3'---CCTAG     G---
                                                                        5'

                                               5'TCGA                   5'---T   CGA---3'
TaqI              Thermus aquaticus
                                               3'AGCT                   3'---AGC   T---5'

                                               5'GANTCA                 5'---G   ANTC---3'
HinfI             Haemophilus influenzae
                                               3'CTNAGT                 3'---CTNA   G---5'

                                               5'GATC                   5'---     GATC---3'
Sau3A             Staphylococcus aureus
                                               3'CTAG                   3'---CTAG     ---5'

                                               5'CAGCTG                 5'---CAG    CTG---3'
PovII*            Proteus vulgaris
                                               3'GTCGAC                 3'---GTC    GAC---5'

                                               5'CCCGGG                 5'---CCC    GGG---3'
SmaI*             Serratia marcescens
                                               3'GGGCCC                 3'---GGG    CCC---5'
                                                                                         54
APPLICATIONS:

 They are used to assist insertion of genes into plasmid
 vectors during gene cloning and protein
 expression experiment.

 Restriction enzymes can also be used to distinguish
 gene alleles by specifically recognizing single base
 changes in DNA known as single nucleotide
 polymorphisms .



                                                        55
Restriction enzyme can be used to genotype a DNA
 sample without the need for expensive gene
 sequencing.

 Restriction enzymes are used to digest genomic DNA
 for gene analysis by Southern blot.




                                                       56
57

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Dhanu

  • 1. 1
  • 3. 3
  • 4.  DNA sequencing is the determination of the order of bases in sample of DNA.  It is the reading of the genetic code.  However, not all DNA sequences are genes (i.e. coding regions) as there may, depending on the organism and the source of the DNA sample, also be promoters, tandem repeats, introns, etc. 4
  • 5. Two methods for the large-scale sequencing of DNA became available in the late 1970's. Both based on generation of DNA fragments of different lengths which start at a fixed point and terminate at specific nucleotides. DNA fragments are separated by size on polyacrylamide gels and the nucleotide sequences are directly read from the gel. 5
  • 6. 1. Maxam-Gilbert sequencing (chemical cleavage method using double-stranded (ds) DNA) in which the sequence of a double-stranded DNA molecule is determined by treatment with chemicals that cut the molecule at specific nucleotide positions. 6
  • 7. 2. Sanger-Coulson sequencing (chain termination method using single-stranded (ss) DNA) in which the sequence of a single-stranded DNA molecule is determined by enzymatic synthesis of complementary polynucleotide chains, these chains terminating at specific nucleotide positions. 7
  • 8. 8
  • 9. Sanger Method of DNA Sequencing Major steps 1. Template DNA (ssDNA) 2. Primer annealing 3. Complementary strand synthesis 4. Labeling for the detection of fragments 5. Chain termination using ddNTPs 6. Resolution on denaturing PAGE 7. Visualization of bands by autoradiography 9
  • 10.  preparation of identical single-stranded DNA molecules. The first step is to anneal a short oligonucleotide to the same position on each molecule, this oligonucleotide subsequently acting as the primer for synthesis of a new DNA strand that is complementary to the template which is to be sequenced . The strand synthesis reaction catalyzed by a DNA polymerase enzyme and requires the four deoxyribonucleotide triphosphates (dNTPs - dATP, dCTP, dGTP and dTTP) as substrates, would normally continue until several thousand nucleotides had been polymerized. 10
  • 11. This does not occur in a chain termination sequencing experiment because, as well as the four dNTPs, a small amount of a dideoxynucleotide (e.g. ddATP) is added to the reaction.  The polymerase enzyme does not discriminate between dNTPs and ddNTPs, so the dideoxynucleotide can be incorporated into the growing chain, but it then blocks further elongation because it lacks the 3′-hydroxyl group needed to form a connection with the next nucleotide 11
  • 12. 12
  • 13. 13
  • 14. 14
  • 15. Reading a DNA Sequencing Gel Sequence 5’ to 3’ C G G G C G T 15
  • 16. The smallest fragments will be at the bottom of the gel, the largest fragments at the top. The DNA sequence can be determined by determining the terminating base for the shortest fragment, then for the next shortest fragment for all of the DNA fragments 16
  • 17. MAXAM-GILBERT SEQUENCING This chemical cleavage method uses double-stranded DNA samples and so does not require cloning of DNA into an M13 phage vector to produce single-stranded DNA.  It involves modification of the bases in DNA followed by chemical base-specific cleavage. Stages: 1. Double-stranded DNA to be sequenced is labeled by attaching a radioactive phosphorus (32P) group to the 5' end. Polynucleotide kinase enzyme and 32P-dATP is used here. 17
  • 18. 2. Using dimethyl sulphoxide (DMSO) and heating to 90oC, the two strands of the DNA are separated and purified (e.g. using gel electrophoresis and the principle that one of the strands is likely to be heavier than the other due to the fact that it contains more purine nucleotides (A and G) than pyrimidines (C and T) which are lighter. 3. Single-stranded sample is split into separate samples and each is treated with one of the cleavage reagents. This part of the process involves alteration of bases (e.g. dimethylsulphate methylates guanine) followed by removal of altered bases. Lastly, piperidine is used for cleavage of the strand at the points where bases are missing 18
  • 19. Chemical Chemical Base Chemical used for used for used for specificity base alteration altered base strand removal cleavage G Dimethylsulphate Piperidine Piperidine A+G Acid Acid Piperidine C+T Hydrazine Piperidine Piperidine Hydrazine + High C Piperidine Piperidine salt A>C Alkali Piperidine Piperidine 19
  • 20. G A+G C +T C Sequence C G T T C C G G A C T A A 20
  • 21. Automated DNA Sequencing with Fluorescent Dyes Each different ddNTP is coupled to a different colored fluorescent dye ddTTP is red; ddGTP is black etc. 21
  • 22. Alternative Sequencing Methods: Pyrosequencing Pyrosequencing is based on the generation of light signal through release of pyrophosphate (PPi) on nucleotide addition. DNAn + dNTP  DNAn+1 + PPI PPi is used to generate ATP from adenosine phosphosulfate (APS). APS + PPI  ATP ATP and luciferase generate light by conversion of luciferin to oxyluciferin. 22
  • 23. Each nucleotide is added in turn.  Only one of four will generate a light signal.  The remaining nucleotides are removed enzymatically.  The light signal is recorded on a pyrogram. DNA sequence: A T C A GG CC T Nucleotide added : A T C A G C T 23
  • 24. Bisulfite Sequencing  Bisulfite sequencing is used to detect methylation in DNA.  Bisulfite deaminates cytosine, making uracil.  Methylated cytosine is not changed by bisulfite treatment.  The bisulfite-treated template is then sequenced. 24
  • 25. Bisulfite Sequencing The sequence of treated and untreated templates is compared. Me Me Me Methylated sequence: GTC GGC GATCTATC GTGCA … Me Me Me Treated sequence: GTC GGC GATUTATC GTGUA … DNA Sequence: (Untreated) reference: ...GTCGGCGATCTATCGTGCA… Treated sequence: ...GTCGGCGATUTATCGTGUA… This sequence indicates that these Cs are methylated. 25
  • 26. Genome sequencing strategies  Only short DNA molecules (~800 bp) can be sequenced in one read, so large DNA molecules, such as genomes, longer sequences must be subdivided into smaller fragments and subsequently reassembled to give the overall sequence.  Genome sequencing can be approached in two ways 26
  • 27. Whole-genome shotgun sequencing The whole-genome shotgun approach was first proposed by Craig Venter and colleagues as a means of speeding up the acquisition of contiguous sequence data for large genomes such as the human genome and those of other eukaryotes (Venter et al., 1998; Marshall 27
  • 28. Clone contig sequencing:  Involves the systematic production and sequencing of sub clones arrange overlapping clones before sequencing. 28
  • 29. DNA MODIFICATION & RESTRICTION Bacteria can destroy an invading or foreign DNA from an other species, thus preventing its replication, transcription, or incorporation in to the host cell genome. This is made possible by an ingenious combination of two enzymatic processes called modification & restriction. 29
  • 31. MODIFICATION  It is the enzymatic alteration of its own DNA by the cell, in a species distinctive way , thus differentiating it from that of other species.  The protective modification of the host cell DNA is brought about by modification methylases,which methylate certain adenine residues.  Once the host cell DNA is modified in this manner,it cannot be degraded by that cells restriction enzymes. 31
  • 32.  The restriction methylases transfer methyl groups from s-adenosylmethionine to pairs of adenine residues in duplex DNA , one in each strand; the two adenine are on adjacent or near by base pairs.  The sequence of bases on the two stands between and near the methylated adenines is symmetrical on either side of a mid point. 32
  • 34. A Restriction Enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restrictionsites. Inside a bacterial host, the restriction enzymes selectively cut up foreign DNA in a process called restrication. To cut the DNA, a restriction enzyme makes two incisions, once through each sugar-phosphate backbone (i.e. each strand) of the DNA double helix. 34
  • 35. 35
  • 36. Restriction Enzymes scan the DNA sequence. 36
  • 37.  recognition site  5'-GTATAC-3'  ::::::  3'-CATATG-5'  A palindromic recognition site reads the same on the reverse strand as it does on the forward strand when both are read in the same orientation.  Restriction enzymes recognize a specific sequence of nucleotides and produce a double-stranded cut in theDNA.  There are two types of palindromic sequences that can be possible in DNA. 37
  • 38. The Mirror like palindrome is similar to those found in ordinary text, in which a sequence reads the same forward and backwards on a single strand of DNA strand, as in GTAATG.  The inverted repeat palindrome is also a sequence that reads the same forward and backwards, but the forward and backward sequences are found in complementary DNA strands (i.e., of double-stranded DNA), as in GTATAC (GTATAC being complementary to CATATG).  Inverted repeat palindromes are more common and have greater biological importance than mirror-like palindromes. 38
  • 39.  Different restriction enzymes that recognize the same sequence are known as neoschizomers.  These often cleave in different locales of the sequence. Different enzymes that recognize and cleave in the same location are known as isoschizomers.  Types  Restriction endonucleases are categorized into three or four general groups (Types I, II and III) based on their composition and enzyme cofactor requirements, the nature of their target sequence, and the position of their DNA cleavage site relative to the target sequence. 39
  • 40.  There are four classes of restriction endonucleases: types I, II,III and IV. All types of enzymes recognise specific short DNA sequences and carry out the endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5'- phosphates  They differ in their recognition sequence, subunit composition, cleavage position, and cofactor requirements 40
  • 41.  Type I restriction enzymes were the first to be identified and were first identified in two different strains (K-12 and B) of E. coli.  These enzymes cut at a site that differs, and is a random distance (at least 1000 bp) away, from their recognition site.  Cleavage at these random sites follows a process of DNA translocation, which shows that these enzymes are also molecular motors.  The recognition site is asymmetrical and is composed of two specific portions—one containing 3–4 nucleotides, and another containing 4–5 nucleotides— separated by a non-specific spacer of about 6–8 nucleotides. 41
  • 42.  These enzymes are multifunctional and are capable of both restriction and modification activities, depending upon the methylation status of the target DNA.  The cofactors S-Adenosyl methionine (AdoMet), hydrolyzed adenosine triphosphate (ATP), and magnesium (Mg2+) ions, are required for their full activity.  Type I restriction enzymes possess three subunits called HsdR, HsdM, and HsdS; 42
  • 43.  HsdR is required for restriction; HsdM is necessary for adding methyl groups to host DNA (methyltransferase activity) and HsdS is important for specificity of the recognition (DNA-binding) site in addition to both restriction (DNA cleavage) and modification (DNA methyltransferase) activity.[ 43
  • 44. Type II: 44
  • 45.  They are a dimer of only one type of subunit; their recognition sites are usually undivided and palindromic and 4–8 nucleotides in length, they recognize and cleave DNA at the same site, and they do not use ATP or AdoMet for their activity—they usually require only Mg2+ as a cofactor.[  These are the most commonly available and used restriction enzymes. 45
  • 46.  In the 1990s and early 2000s, new enzymes from this family were discovered that did not follow all the classical criteria of this enzyme class, and new subfamily nomenclature was developed to divide this large family into subcategories based on deviations from typical characteristics of type II enzymes. 46
  • 47.  Type IIB restriction enzymes (e.g. BcgI and BplI) are multimers, containing more than one subunit  They cleave DNA on both sides of their recognition to cut out the recognition site.  They require both AdoMet and Mg2+ cofactors. Type IIE restriction endonucleases (e.g. NaeI) cleave DNA following interaction with two copies of their recognition sequence.[  One recognition site acts as the target for cleavage, while the other acts as an allosteric effector that speeds up or improves the efficiency of enzyme cleavage. 47
  • 48.  Type IIG restriction endonucleases (Eco57I) do have a single subunit, like classical Type II restriction enzymes, but require the cofactor AdoMet to be active.  Type IIM restriction endonucleases, such as DpnI, are able to recognize and cut methylated DNA  Type IIS restriction endonucleases (e.g. FokI) cleave DNA at a defined distance from their non-palindromic asymmetric recognition sites  These enzymes may function as dimers. Similarly, Type IIT restriction enzymes (e.g., Bpu10I and BslI) are composed of two different subunits 48
  • 49.  Type III restriction enzymes (e.g. EcoP15) recognize two separate non-palindromic sequences that are inversely oriented. They cut DNA about 20-30 base pairs after the recognition site.  These enzymes contain more than one subunit and require AdoMet and ATP cofactors for their roles in DNA methylation and restriction, respectively.  They are components of prokaryotic DNA restriction- modification mechanisms that protect the organism against invading foreign DNA.  49
  • 50.  Type III enzymes are hetero- oligomeric, multifunctional proteins composed of two subunits, Res and Mod.  The Mod subunit recognises the DNA sequence specific for the system and is a modification methyltransferase; as such it is functionally equivalent to the M and S subunits of type I restriction endonuclease.  Res is required for restriction, although it has no enzymatic activity on its own. 50
  • 51.  Type III enzymes recognise short 5-6 bp long asymmetric DNA sequences and cleave 25-27 bp downstream to leave short, single-stranded 5' protrusions  They require the presence of two inversely oriented unmethylated recognition sites for restriction to occur. These enzymes methylate only one strand of the DNA, at the N-6 position of adenosyl residues, so newly replicated DNA will have only one strand methylated, which is sufficient to protect against restriction. 51
  • 52.  Type III enzymes belong to the beta-subfamily of N6 adenine methyltransferases, containing the nine motifs that characterize this family, including motif I, the AdoMet binding pocket (FXGXG), and motif IV, the catalytic region (S/D/N (PP) Y/F).[ 52
  • 53. 5'GGTACC 5'---GGTAC C---3' Klebsiella pneumoniae 3'CCATGG 3'---C CATGG---5' 5'CTGCAG 5'---CTGCA G---3' PstI[48] Providencia stuartii 3'GACGTC 3'---G ACGTC---5' Streptomyces 5'GAGCTC 5'---GAGCT C---3' SacI[48] achromogenes 3'CTCGAG 3'---C TCGAG---5' 5'GTCGAC 5'---G TCGAC---3' SalI[48] Streptomyces albus 3'CAGCTG 3'---CAGCT G---5' Streptomyces 5'AGTACT 5'---AGT ACT---3' ScaI[48] caespitosus 3'TCATGA 3'---TCA TGA---5' 5'ACTAGT 5'---A CTAGT---3' SpeI Sphaerotilus natans 3'TGATCA 3'---TGATC A---5' Streptomyces 5'GCATGC 5'---GCATG C---3' SphI[48] phaeochromogenes 3'CGTACG 3'---C GTACG---5' Streptomyces 5'AGGCCT 5'---AGG CCT---3' StuI[49][50] tubercidicus 3'TCCGGA 3'---TCC GGA---5' 5'TCTAGA 5'---T CTAGA---3' 53 XbaI[48] Xanthomonas badrii
  • 54. Enzyme Source Recognition Sequence Cut 5'---G AATTC--- 5'GAATTC 3' EcoRI Escherichia coli 3'CTTAAG 3'---CTTAA G--- 5' 5'CCWGG 5'--- CCWGG---3' EcoRII Escherichia coli 3'GGWCC 3'---GGWCC ---5' 5'---G GATCC--- 5'GGATCC 3' BamHI Bacillus amyloliquefaciens 3'CCTAGG 3'---CCTAG G--- 5' 5'TCGA 5'---T CGA---3' TaqI Thermus aquaticus 3'AGCT 3'---AGC T---5' 5'GANTCA 5'---G ANTC---3' HinfI Haemophilus influenzae 3'CTNAGT 3'---CTNA G---5' 5'GATC 5'--- GATC---3' Sau3A Staphylococcus aureus 3'CTAG 3'---CTAG ---5' 5'CAGCTG 5'---CAG CTG---3' PovII* Proteus vulgaris 3'GTCGAC 3'---GTC GAC---5' 5'CCCGGG 5'---CCC GGG---3' SmaI* Serratia marcescens 3'GGGCCC 3'---GGG CCC---5' 54
  • 55. APPLICATIONS:  They are used to assist insertion of genes into plasmid vectors during gene cloning and protein expression experiment.  Restriction enzymes can also be used to distinguish gene alleles by specifically recognizing single base changes in DNA known as single nucleotide polymorphisms . 55
  • 56. Restriction enzyme can be used to genotype a DNA sample without the need for expensive gene sequencing.  Restriction enzymes are used to digest genomic DNA for gene analysis by Southern blot. 56
  • 57. 57