DNA SEQUENCING METHODS AND STRATEGIES FOR GENOME SEQUENCING

PresentedBy:-
Neeraj Chaturvedi
Nikhat Fatima
Kaynat Arif
Kirtika Adhikari
Puneet Kulyana
1

 DNA sequencing is the process of determining
the precise order of nucleotides within
a DNA molecule.
 It includes any method or technology that is
used to determine the order of the four bases—
Adenine, Guanine, Cytosine, and Thymine—in
a strand of DNA 2

 Basic methods
o Maxam-Gilbert sequencing
o Chain-termination methods
 Advanced methods and de novo sequencing
o Shotgun sequencing
o Bridge PCR
 High-throughput methods
3

 Allan Maxam and Walter Gilbert published a DNA sequencing method in 1977 based on
chemical modification of DNA and subsequent cleavage at specific bases.
 It was the first widely adopted method for DNA sequencing, and, along with the Sanger
dideoxy method, represents the first generation of DNA sequencing methods.
4

 Radioactive labeling is done at one 5′ end of the DNA fragment to be sequenced.
 Chemical treatment generates breaks at a small proportion of one or two of the four
nucleotide bases in each of four reactions.
 The modified DNAs may then be cleaved.
 Thus a series of labeled fragments is generated, which are then electrophoresed side by side
in denaturing acrylamide gels for size separation
To visualize the fragments, the gel is exposed to X-ray film
5

 Sanger’s method is also known as dideoxy chain termination method. It was developed by
the British biochemist Fred Sanger and his colleagues in 1977.
 PRINCIPLE:
The Sanger Technique uses dideoxynucleotides (dideoxyadenine, dideoxyguanine,
etc) These are molecules that resemble normal nucleotides but lack the normal -OH group.
 Because they lack the -OH (which allows nucleotides to join a growing DNA strand),
replication stops.
7

1. Template preparation:-
 Copies of template strand to be sequenced must be prepared with short known
sequences at 3’ end of the template strand.
2. Generation of nested set of labelled fragments:-
 Copies of each template is divided into four batches and each batch is used for
different replication reaction.
 To synthesize fragments that terminates at A, ddATP is added to the reaction mixture
on batch I along with dATP, dTTP, dCTP and dGTP, standard primer and DNA
polymerase I.
8

3. Electrophoresis:-
 The reaction mixture from four batches are
loaded into four different well on
polyacrylamide gel and electrophoresed.
 The autoradiogram of the gel is read to
determine the order of bases of
complementary strand to that of template
strand.
4. Gel Separation:-
 The reaction mixtures are separated on a
denaturing polyacrylamide gel.
 Polyacrylamide can separate the DNA
strands which differ in length by only one
nucleotide.
 The separated neucleotides are separated
by radioactive method or fluorescent
method.
10

 Molecular biology:- To study genomes and the proteins they encode.
 Evolutionary biology:- Is used in evolutionary biology to study how different organisms are
related and how they evolved.
 Metagenomics:- It involves identification of organisms present in a body of water, sewage, dirt,
debris filtered from the air, or swab samples from organisms. Knowing which organisms are
present in a particular environment is critical to research.
 Medicine:- Medical technicians may sequence genes (or, theoretically, full genomes) from
patients to determine if there is risk of genetic diseases.
 Forensics:- DNA sequencing may be used along with DNA profiling methods for forensic
identification[4] and paternity testing.
11

 Term “genome” was coined by H. Winkler in 1920
 A genome can be defined as the entire DNA content of each nucleated cell in an organism
 Humans for example have a genome that is encoded on 46 chromosomes, organized into
23 pairs, out of which 44 chromosomes are autosomes and 2 are sex chromosomes
 Each genome contains all of the information needed to build that organism and allow it
to grow and develop
 Genome is a unique sequence of DNA
 It is over 3 billion letters long and is found in almost every cell in the body
12

 Genome sequencing is the technique that allows researchers to read the genetic
information found in the DNA of anything from bacteria to plants to animals
 Sequencing involves determining the order of bases, the nucleotide subunits-
Adenine(A), Guanine(G), Cytosine(C) and Thymine(T), found in DNA
 It has largely been used as a research tool, but is currently being introduces to clinics
 Genome sequencing should not be confused with DNA profiling, which only
determines the likelihood that genetic material came from a particular individual or
group and doesn’t contain information on origin or susceptibility to specific diseases
 Sequencing a genome is an enormous task. It requires not only finding the nucleotide
sequence of small pieces of genome but also ordering those small pieces together into
the whole genome.
13

 Break genome into smaller fragments
 Sequence those smaller pieces
 Place the sequences of the short fragments
together
14

 Two different methods used :-
1) Hierarchical shotgun sequencing
– useful for sequencing genomes of higher vertebrates that contain
repetitive sequences
2) Whole genome shotgun sequencing
– useful for smaller genome
 Factors that determine sequencing strategy are :-
a) Genome size
b) Chromosomal structure
c) Repeat content and character
d) Desired end product
15

 The method preferred by the HGP is the hierarchical shotgun sequencing method.
 Also known as
- The Clone-by-clone strategy
- The map-based method
- map first, sequence later
- top-down sequencing
 In clone based sequencing the first step is mapping.
 One first constructs a map of the chromosome, making them at regular intervals of
about 100 kilo bases. Then known segments of marked chromosome are cloned in
plasmids.
 One special type of plasmid used for genome sequencing is BAC, which contain DNA
fragment of about 80-180 kb in E.Coli cells.
 The plasmid cells are then further broken into small, random, overlapping fragments of
0.5 to 1.0 kb.
 Finally automated sequencing machines determine the order of each nucleotide of the
small fragments.
The National Human Genome Research Institute used clone based sequencing for the
human genome, for this they relied on computer scientists to assemble the final sequence
16

 J. Craig Venter and H. Smith developed “whole genome shotgun”
sequencing and sequenced the genome of bacteria H. Influenza and M.
genitalicum .
 This approach maybe characterized into 4 steps :-
1) Library construction : the chromosome is isolated from the desired
cells following the methods of molecular biology and randomly
fragments into small pieces using ultrasonic waves. Then the
fragments are purified and attached to the plasmid vector. Plasmids
with single insert are isolated. A library of plasmid clone is prepared
transforming E. Coli strains with plasmid lacked restriction
enzymes.
2) Random sequencing : the DNA is purified from the plasmid.
Thousands of DNA fragments are sequenced using automated
sequencer.
3) Fragment alignment and gap closure : by using special computer
programmes, the sequenced DNA fragments are clustered. Two
fragments are joined together to form large stretch of DNA
4) Proof reading : proof reading is done carefully so that any
ambiguities could be resolved. The sequence is also checked for
frameshift mutation if so, the mutation is corrected.
17

 In this, sequence reads are generated in
both a clone-by-clone and a whole genome
fashion.
 A hybrid shotgun- sequencing strategy
can, in principle, capture the
advantageous elements of both clone-by-
clone and whole-genome approaches.
 For eg. The whole-genome shotgun
component provides the rapid insight
about the sequence of the entire genome.
18

 Next-generation sequencing (NGS), also known as high-
throughput sequencing, is the catch-all term used to describe
a number of different modern sequencing technologies
including:
1) Illumina (Solexa) sequencing
2) Roche 454 sequencing
3) Ion torrent: Proton / PGM sequencing
4) SOLiD sequencing
 These recent technologies allow us to sequence DNA and
RNA much more quickly and cheaply than the previously
used Sanger sequencing, and as such have revolutionised the
study of genomics and molecular biology.
19

ADVANTAGES DISADVANTAGES
1) Normalized coverage of all
genes
2) Information about gene
structure
3) Information about regulatory
elements
4) Genome organization
1) High cost
2) Time consuming
3) Difficult to determine if a
sequence codes for a gene
20

 Gene expression is a highly regulated mechanism that controls the function and
adaptability of all living cells including prokaryotes and eukaryotes.
 Several techniques exist for studying and quantifying gene expression and its regulation.
 Some of these techniques are old and well established while others are relatively new,
multiplex techniques.
 The field of gene expression analysis has undergone major advances in biomedical
research.
 Traditional methods focused on measuring the expression of one gene at a time and not
in any particular biological context.
 However, today, mRNA expression techniques have led to improvements in gene
identification and disease sub-classification, for example.
22

 SAGE
 Electrophoresis
 Microarray
 Western Blotting
 Proteomics
 Immunohistochemistry
23

 SAGE is a technique used to create a library of short sequence tags which can
each be used to detect a transcript.
 The expression level of the transcript can be determined by assessing how many
times each tag is detected.
 This technology enables comprehensive expression analysis across the genome.
 A 9 bp tag is sufficient to unambiguously identify a gene
 Concatenation (linking together) of these short DNA sequences increases the
efficiency of identifying unique transcripts in a serial manner.
24

1. mRNA to cDNA
2. Cleave with A.E
3. Isolate 3’ most transcript of each cDNA by binding
to Streptavidin beads
4. Divide cDNA in half
5. Ligate to 1 of 2 linkers (each with a T.E site)
6. Ligate the two pools of tags together….
25

7. Ligated linkers serve as primers for
amplification
8. Cleave PCR products with A.E. to
isolate ditags
9. Concatenate by ligation
10. Clone
11. Sequence
26

 Gene Discovery
 Analysis of Cardiovascular gene expression
 Gene expression in carcinogenesis
 Substance abuse studies
 Cell, tissue and developmental stage profiles in C. elegans
 Profiling of human diseases and more…..
27

 A technique for detecting specific proteins separated
by electrophoresis by use of labeled antibodies. So
called since it has some similarity to a Southern blot.
 The Western Blot is an analytical technique used to
detect specific proteins in a given sample of tissue
homogenate or extract.
 Western blotting is a technique for detecting specific
protein molecules within a protein mixture.
 This mixture might include all the proteins that are
associated with a certain cell type or tissue.
 The technique can help to determine a protein’s
size, and how much of it is expressed.
28

 Western blot analysis can analyze any
protein sample whether from cells or
tissues, but also can analyze recombinant
proteins synthesized in vitro.
 Western blot is dependent on the quality
of antibody you use to probe for your
protein of interest, and how specific it is
for this protein.
29

 Tissue preparation
 Gel electrophoresis
 Transfer
 Blocking
 Detection
 Analysis
30

 Samples may be taken from whole tissue or from cell culture. In most cases, solid
tissues are first broken down mechanically using a blender.
 It should be noted that bacteria, virus or environmental samples can be the
source of protein and thus Western blotting is not restricted to cellular studies
only.
 Assorted detergents, salts, and buffers may be employed to encourage lysis of
cells and to solubilize proteins.
 Tissue preparation is often done at cold temperatures to avoid protein
denaturing.
31

 The proteins of the sample are separated using gel electrophoresis. Separation of
proteins may be by isoelectric point molecular weight, electric charge, or a
combination of these factors.
 The principle involved is the difference in the ELECTROPHORETIC
MOBILITIES of different proteins.
32

 In order to make the proteins accessible to antibody detection, they are moved
from within the gel onto a membrane made of nitrocellulose or polyvinylidene
difluoride (PVDF).
 The membrane is placed on top of the gel, and a stack of filter papers placed on
top of that. The entire stack is placed in a buffer solution which moves up the
paper by capillary action, bringing the proteins with it.
 Another method for transferring the proteins is called electro blotting and uses an
electric current to pull proteins from the gel into the PVDF or nitrocellulose
membrane.
33

 The membrane has the ability to bind to proteins in in this case both the target
and antibodies are proteins and so there could be some unwanted binding.
 Blocking of non-specific binding is achieved by placing the membrane in a dilute
solution of protein - typically Bovine serum albumin(BSA) with a minute
percentage of detergent such as Tween 20.
 The protein in the dilute solution attaches to the membrane in all places where
the target proteins have not attached. Thus, when the antibody is added, there is
no room on the membrane for it to attach other than on the binding sites of the
specific target protein. Blocking Presented by Pri
34

 During the detection process, the
membrane is "probed" for the protein
of interest with a modified antibody
which is linked to a reporter enzyme,
which when exposed to an appropriate
substrate drives a colorimetric
reaction and produces a color.
35

 After the unbound probes are washed away, the western blot is ready for
detection of the probes that are labeled and bound to the protein of interest.
 Size approximations are taken by comparing the stained bands to that of the
marker loaded during electrophoresis.
 The process is repeated for a structural protein, such as actin or tubulin that
should not change between samples.
36

 While ELISA being a non
specific test, Western blotting is
a more specific test for detection
of HIV. It can detect one protein
in a mixture of proteins while
giving information about the
size of the protein and so is
more specific.
 Western blot test is referred to
as the 'Gold Standard’
 It also tells you how much
protein has accumulated in
cells. 37
DISADVANTA
GES If a protein is degraded quickly,
Western blotting won't detect it well
 This test takes longer that other
existing tests
 It might also be more costly

 Functional genomics involves the analysis of large datasets of information derived
from various biological experiments.
 One such type of large-scale experiment involves monitoring the expression levels of
thousands of genes simultaneously under a particular condition, called gene expression
analysis.
 Microarray technology makes this possible and the quantity of data generated from
each experiment is enormous, dwarfing the amount of data generated by genome
sequencing projects.
 All of our cells carry some amount and type of genes
 Expression varies from type of cells(normal vs tumour), from time to time (different
development stages), & environmental response
 Microarray is used to detect DNA, protein, RNA, antibodies etc.
 Most commonly used DNA microarray
38

 It measures amount of mRNA in a given cell at a particular time
 It is divided into 2 stages:
STAGE 1 :- produce DNA chip:
- extract DNA
- amplify using PCR
- DNA denaturation and get ssDNA
STAGE 2 :- transcriptome:
- total mRNA extract at a particular time from a particular cell
- reverse transcribe and produce cDNA using reverse transcriptase
This cDNA works as control and tagged with cy3 dye(green)
39

 There are various applications of DNA microarray. Some of them are:-
1) To check which gene is expressed in a particular cell and condition
2) To evaluate which therapy is best
3) Which specific genes are affected in a variety of disease (heart disease, diabetes,
cancer etc.)
4) Classification of disease
40

REFERENCES
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3831009/
www.ebi.ac.uk/.../what-next-generation-dna-
https://www.ncbi.nlm.nih.gov/pubmed/20486139http://
www.cell.com/molecular-cell/abstract/S1097-2765(15)00340-8
www.google.co.in
41

DNA SEQUENCING METHODS AND STRATEGIES FOR GENOME SEQUENCING

More Related Content

What's hot (20)

Similar to DNA SEQUENCING METHODS AND STRATEGIES FOR GENOME SEQUENCING (20)

Recently uploaded (20)

DNA SEQUENCING METHODS AND STRATEGIES FOR GENOME SEQUENCING