DNA sequencing determines the order of bases in a DNA sample. Two major early methods are the Maxam-Gilbert chemical cleavage method using double-stranded DNA and the Sanger chain termination method using single-stranded DNA. The Sanger method involves primer annealing, complementary strand synthesis using labeled chain terminators, and resolution on a polyacrylamide gel to read the sequence. Bacteria protect their DNA from foreign DNA through modification and restriction. Modification involves host-specific methylation while restriction enzymes cut foreign DNA at specific recognition sequences. There are four classes of restriction enzymes that differ in subunit composition, cofactor requirements, and cleavage position relative to the recognition site.
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
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
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
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
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
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