SEQUENCING

• Sequence Analysis is the process of subjecting a DNA, RNA or peptide

sequence to any of a wide range of analytical methods to understand
its features, function, structure, or evolution
• DNA sequencing is the process of determining the sequence of
nucleotide bases (As, Ts, Cs, and Gs) in a piece of DNA.
• Today, with the right equipment and materials, sequencing a short
piece of DNA is relatively straightforward.
METHODS OF DNA SEQUENCING

• Historically , there are two main methods

1. Maxam Gilbert method or Chemical cleavage method
2. Sangers method or chain termination method

Some other sequencing methods

3. Automated sequencing
4. Whole genome sequencing
5. Next generation sequencing
 MAXAM GILBERT METHOD OR CHEMICAL CLEAVAGE METHOD
• By A.M. Maxam and W.Gilbert-1977
• Chemical sequencing Treatment of DNA with certain chemicals
• DNA cuts into fragments to monitor the sequences.
• PRINCIPLE
The partially cleaved DNA fragment is subjected to five separate chemical
reactions.
Each of which is specific for a particular base.
The resulting fragment terminate at that specific base followed by high
resolution gel electrophoresis and detection of the labelled fragments by
autoradiography.
• METHOD
• Labelled DNA at one end with 32P.
• DNA copies are divided in to 4 samples.
• Each samples treated with a chemical that specifically destroys one or two of
the 4 base in DNA.
• Chemical treatment generates breaks at a small proportion of one or two of
the four nucleotide bases in each of four reactions (G, A+G, C, C+T). For
example, the purines (A+G) are depurinated using formic acid, the guanines
(and to some extent the adenines) are methylated by dimethyl sulfate, and
the pyrimidines (C+T) are hydrolysed using hydrazine.
• The addition of salt (sodium chloride) to the hydrazine reaction inhibits the
reaction of thymine for the C-only reaction. The modified DNAs may then be
cleaved by hot piperidine; (CH2)5NH at the position of the modified base.
• The concentration of the modifying chemicals is controlled to introduce
on average one modification per DNA molecule. Thus a series of labelled
fragments is generated, from the radiolabelled end to the first "cut" site
in each molecule.

• The fragments in the four reactions are electrophoresed side by side in

denaturing acrylamide gels for size separation. To visualize the fragments,
the gel is exposed to X-ray film for autoradiography, yielding a series of
dark bands each showing the location of identical radiolabelled DNA
molecules. From presence and absence of certain fragments the
sequence may be inferred.
 SANGER SEQUENCING
INTRODUCTION
• The DNA sequencing method developed by Fred Sanger forms the basis of
automated "cycle" sequencing reactions today.
• In the 1980s, two key developments allowed researchers to believe that
sequencing the entire genome could be possible.
-The first was a technique called polymerase chain reaction (PCR) that
enabled many copies of DNA sequence to be quickly and accurately
produced.
-The second, an automated method of DNA sequencing, built upon the
chemistry of PCR and the sequencing process developed by Frederick
Sanger in 1977.
• Sanger sequencing reaction also contains a unique ingredient:
- Dideoxy, or chain-terminating, versions of all four nucleotides (ddATP,
ddTTP, ddCTP, ddGTP), each labelled with a different colour of dye.

• Dideoxy nucleotides are similar to regular, or deoxy, nucleotides, but with

one key difference: they lack a hydroxyl group on the 3’ carbon of the
sugar ring.

• In a regular nucleotide, the 3’ hydroxyl group acts as a “hook," allowing a

new nucleotide to be added to an existing chain.
• PRINCIPLE
The 5’ carbon of an “incoming” deoxynucleotide (dNTP) is joined to the 3’
carbon at the end of the chain. Hydroxyl groups in each position form ester
linkages with a central phosphate. In this way, the nucleotide chain elongates.
• The key to Sanger’s sequencing method is the peculiar chemistry of
dideoxynucleotides (ddNTP). Like a deoxynucleotide, a ddNTP is incorporated
into a chain by forming a phosphodiester linkage at its 5’ end.
• However, the ddNTP lacks a 3’ hydroxyl group (OH) necessary to form the
linkage with an incoming nucleotide. So the addition of a ddNTP halts
elongation.
• METHOD
• To sequence DNA, four separate reactions are necessary ; one to provide
sequence information about each of the nucleotides. Each reaction contains:
template DNA, a short primer (about 20 nucleotides), DNA polymerase, and
the four dNTPs (one radioactively labelled).
• One type of ddNTP – A, T, C, or G – is added to each.
• The mixture is first heated to denature the template DNA (separate the
strands), then cooled so that the primer can bind to the single- stranded
template.
• The DNA Polymerase makes no distinction between dNTPs or ddNTPs. Each
time a ddNTP is incorporated, in this case ddATP, synthesis is “terminated”
and a DNA strand of a discrete size is generated. In this sequencing example,
the ddATP (purple) has terminated the reaction. The dATP happens to be the
radioactive tracer, but this has no effect on elongation.
• Because billions of DNA molecules are present, the elongation reaction can
be terminated at any adenine position. This results in collections of DNA
strands of different lengths. The same is true for the other three terminator
reactions.
• Each reaction is then loaded into a separate lane of a polyacrylamide gel
containing urea, which prevents the DNA strands from renaturing during
electrophoresis.
• Ionized phosphates give the DNA molecule a negative charge, so DNA
migrate toward the positive pole of an electric field. The movement of DNA
molecules through the polyacrylamide matrix is size dependent.
• Over the course of electrophoresis, shorter DNA molecules will move further
down the gel than larger ones. Millions of terminated molecules of the same
size will migrate to the same place and “band” in the gel.
• After electrophoresis, the gel is sandwiched against X-ray film. The radioactive
adenine in the synthesized DNA emit beta particles that expose the film,
making a record of the positions of DNA bands in the gel.
• The sequencing gel is then read from bottom to top. The sequence of bands in
the various terminator lanes gives the sequence of nucleotides in the template
DNA.
• USES AND LIMITATIONS
• Sanger sequencing gives high-quality sequence for relatively long
stretches of DNA (up to about 900 base pairs).
• It's typically used to sequence individual pieces of DNA, such as bacterial
plasmids or DNA copied in PCR.

• However, Sanger sequencing is expensive and inefficient for larger-scale

projects, such as the sequencing of an entire genome. For tasks such as
these, new, large-scale sequencing techniques are faster and less
expensive.
AUTOMATED SEQUENCING
• Florescence technique is used for detection of DNA bands.

• Fluorescent labelled dd NTP are used.

• Resulting DNA strands are separated in 4 different lane in

electrophoresis.

• Detected by fluorescence detector.

• Automated DNA sequencing involves four fluorophores, one for each of the four nucleotide
bases.
• The resulting fluorescent signal is recorded at a fixed point when DNA passes through a capillary
containing an electrophoretic gel.
• The base-specific fluorescent labels are attached to appropriate dideoxynucleotide
triphosphates (ddNTP). Each ddNTP is labeled with a different color, e.g., ddATP green, ddCTP
blue, ddGTP yellow, and ddTTP red. (The actual colors for each nucleotide may be different.)
• All chains terminated at an adenine (A) will yield a green signal; all chains terminated at a
cytosine (C) will yield a blue signal, and so on.
• The sequencing reactions based on this kind of chain termination at labelled nucleotides are
carried out automatically in sequencing capillaries.
• The electrophoretic migration of the ddNTP-labelled chains in the gel in the capillary pass in
front of a laser beam focused on a fixed position. The laser induces a fluorescent signal that is
dependent on the specific label representing one of the four nucleotides.
• The sequence is electronically read and recorded and is visualized as alternating peaks in one of
the four colours, representing the alternating nucleotides in their sequence positions.
• In practice the peaks do not necessarily show the same maximal intensity as in the schematic
diagram shown here.
 NEXT GENERATION SEQUENCING
• The principle behind Next Generation Sequencing (NGS) is similar to that
of Sanger sequencing, which relies on capillary electrophoresis.
• The genomic strand is fragmented, and the bases in each fragment are
identified by emitted signals when the fragments are ligated against a
template strand.
• The NGS method uses array-based sequencing which combines the
techniques developed in Sanger sequencing to process millions of
reactions in parallel, resulting in very high speed and throughput at a
reduced cost.
• Three general steps in NGS
1. Library preparation: libraries are created using random fragmentation
of DNA, followed by ligation with custom linkers
2. Amplification: the library is amplified using clonal amplification methods
and PCR
3. Sequencing: DNA is sequenced using one of several different
approaches.
• LIBRARY PREPARATION
• Firstly, DNA is fragmented either enzymatically or by sonication (excitation
using ultrasound) to create smaller strands.
• Adaptors (short, double-stranded pieces of synthetic DNA) are then ligated to
these fragments with the help of DNA ligase, an enzyme that joins DNA strands.
• The adaptors enable the sequence to become bound to a complementary
counterpart.
• Adaptors are synthesized so that one end is 'sticky' whilst the other is 'blunt'
(non-cohesive) with the view to joining the blunt end to the blunt ended DNA.
This could lead to the potential problem of base pairing between molecules
and therefore dimer formation. To prevent this, the chemical structure of DNA
is utilised, since ligation takes place between the 3′-OH and 5′-P ends. By
removing the phosphate from the sticky end of the adaptor and therefore
creating a 5′-OH end instead, the DNA ligase is unable to form a bridge
between the two termini.
• In order for sequencing to be successful, the library fragments need to be
spatially clustered in PCR colonies or ‘colonies' as they are conventionally
known, which consist of many copies of a particular library fragment.
• Since these colonies are attached in a planar fashion, the features of the
array can be manipulated enzymatically in parallel.
• This method of library construction is much faster than the previous labour
intensive procedure of colony picking
• AMPLIFICATION
• Library amplification is required so that the received signal from the
sequencer is strong enough to be detected accurately.

• With enzymatic amplification, phenomena such as 'biasing' and

'duplication' can occur leading to preferential amplification of certain
library fragments.

• Instead, there are several types of amplification process which use PCR to
create large numbers of DNA clusters.
• Emulsion PCR
• Emulsion oil, beads, PCR mix and the library DNA are mixed to form an
emulsion which leads to the formation of micro wells.
• In order for the sequencing process to be successful, each micro well should contain one
bead with one strand of DNA (approximately 15% of micro wells are of this composition).
• The PCR then denatures the library fragment leading two separate strands, one of which
(the reverse strand) anneals to the bead.
• The annealed DNA is amplified by polymerase starting from the bead towards the primer
site.
• The original reverse strand then denatures and is released from the bead only to re-anneal
to the bead to give two separate strands. These are both amplified to give two DNA strands
attached to the bead.
• The process is then repeated over 30-60 cycles leading to clusters of DNA. This technique
has been criticised for its time consuming nature, since it requires many steps (forming and
breaking the emulsion, PCR amplification, enrichment etc) despite its extensive use in many
of the NGS platforms.
• It is also relatively inefficient since only around two thirds of the emulsion micro reactors
will actually contain one bead.
• Therefore an extra step is required to separate empty systems leading to more potential
inaccuracies
• BRIDGE PCR
• The surface of the flow cell is densely coated with primers that are
complementary to the primers attached to the DNA library fragments.
• The DNA is then attached to the surface of the cell at random where it is exposed
to reagents for polymerase based extension.
• On addition of nucleotides and enzymes, the free ends of the single strands of
DNA attach themselves to the surface of the cell via complementary primers,
creating bridged structures.
• Enzymes then interact with the bridges to make them double stranded, so that
when the denaturation occurs, two single stranded DNA fragments are attached
to the surface in close proximity.
• Repetition of this process leads to clonal clusters of localised identical strands.
• In order to optimise cluster density, concentrations of reagents must be
monitored very closely to avoid overcrowding.
 APPLICATIONS OF NEXT GEN
SEQUENCING
• Next generation sequencing has enabled researchers to collect vast
quantities of genomic sequencing data.
• This technology applications include: diagnosing and understanding complex
diseases; whole-genome sequencing; analysis of epigenetic modifications;
transcriptome sequencing − understanding how altered expression of
genetic variants affects an organism; and exome sequencing − mutations in
the exome are thought to contain up to 90% of mutations in the human
genome, which leads to disease.
• DNA techniques have been used to identify and isolate genes responsible for
certain diseases, and provide the correct copy of the defective gene known
as ‘gene therapy’.
Sequence Assembly
Sequence assembly refers to the reconstruction of a DNA
sequence by aligning and merging small DNA fragments.
It is an integral part of modern DNA sequencing.

(1) cutting the DNA into small pieces

(2) reading the small fragments
(3) reconstituting the original DNA by merging the
information on various fragment
Sequence Alignment
• Sequence Alignment is a way of arranging the
sequences of DNA, RNA, or protein to identify
regions of similarity that may be a consequence of
functional, structural or evolutionary relationships
between the sequences.

• It involves the identification of the correct

location of deletions and insertions that
have occurred in either of the two lineages
since the divergence from a common ancestor
TYPES
• Based on number of comparing
sequencing strand, there are two types:

Pair-wise Alignment

Multiple Sequence Alignment

Pair-wise Sequence Alignment
• Pair-wise sequence alignment only compares
two sequences at a time.
• Optimality is based on SCORE.

A pairwise alignment consists of a series of paired

bases, one base from each sequence. There are
three types of pairs:

(1) matches = the same nucleotide appears in

both sequences.
(2) mismatches = different nucleotides are found
in the two sequences.
(3) gaps = a base in one sequence and a null base
in the other.
DATABASES
• Biological databases are libraries of life
sciences information, collected from scientific
experiments, published literature, high throughput
experiment technology, and computational analysis.

 UniGene is an NCBI database of the TRANCRIPTOME

 DNA Data Bank of Japan (DDBJ)
 European Bioinformatics Institute (EMBLEBI)
 SWISS-PROT &Tr-EMBL