description about DNA sequencing techniques

1. PROF S.SUBBIAH et al.
PROF.S.SUBBIAH et al.
DNA sequencing
Department of Surgical Oncology
Centre for Oncology
GRH,Royapettah

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1869 - Fiedrich Miescher – discovered & isolated
DNA
1953- Watson & Crick proposed Double helical
model of DNA
1970- Location-specific primer extension strategy
was employed by Ray wu
1972- Walter Fiers - RNA sequencing of complete
genome of bacteriophage
1977- Fredrick Sanger – Sangers
1977- Gilbert & Maxam – Sequencing by Chemical
degradation

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• DNA sequencing is a process of determining or identifying the order
of nucleotides present in a DNA sequence
• Basic methods - chemical degradation and chain termination - tedious
and time-consuming – lack of automation
• The first semi-automated DNA method was developed
by Lorey and Smith in the year 1986.
• Next generation sequencing

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What are the steps in DNA sequencing?
• Sample preparation (DNA extraction)
• PCR amplification of target sequence
• Amplicons purification
• Sequencing pre-prep
• DNA Sequencing
• Data analysis

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Sample preparation
• Extraction of the DNA
• Cells are treated with detergents, surfactants, proteases, RNAses to break the
cell membranes and other intracellular organelles
• Treated with concentrated saline to cause clumping of protein, lipids and RNA
debris
• Centrifugation to separate debris from DNA
• DNA purification by ethanol or isopropyl alcohol

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PCR amplification of target DNA
• Target - single gene - Gene of interest is isolated and the rest of
the DNA is discarded.
• Flanking primers are added that anneal at the outer regions of
the DNA sequence of interest - unwanted DNA can’t amplify
• Target - whole genome - whole genome is fragmented into pieces
of variable length and fed into PCR machine
• Standard PCR conditions - 35 cycles, denaturation at 94°C,
annealing at 55°C to 65°C and extension at 72°C.

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Amplicon purification
• Unbound primers, primer-dimers, unused Taq DNA polymerase,
unused DNA templates, and other unused PCR buffer components
• Can abort the sequencing
• Spin column-based nucleic acid purification method

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Sequencing pre-preparation
• Primers, nucleotides, High fidelity DNA polymerase
• Primer end - the DNA synthesis
• Amplification process of sequencing
• Similar to PCR, however, here the nucleotides are either radio
or fluorescent-labeled.
• During, the reaction in the sequencer, denaturation, annealing,
and extension occurs, simultaneously.

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DNA sequencing
• Here, as we are using the labeled nucleotides, signals are
produced during the reaction
• The signals of the addition of each complementary nucleotide
are recorded by the machine and the data is sent to the
computer.
• The sequence data is compared with other available data by the
software to find out variations and other mutations present in a
gene

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Different methods of DNA sequencing:
• Maxam and Gilbert method
• Chain termination method
• Semiautomated method
• Automated method
• Pyrosequencing
• The whole-genome shotgun sequencing method
• Clone by the clone sequencing method
• Next-generation sequencing method

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Maxam and Gilbert method
• 1977 - chemical cleavage method
• Chemical modifications of the DNA - further cleavage -
electrophoresis
• Direct sequencing of purified DNA, without requiring
previous in vivo cloning and ssDNA preparation steps.
• Radioactive labeling of the 5′-P ends of double-stranded DNA
(dsDNA) with 32P-dATP
• Denatured with - Hydrazine: T + C, Hydrazine NaCl: C, Dimethyl
sulfate: A + G, Piperidine: G
• resulting ssDNA molecules are segregated via electrophoresis

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• No prior knowledge of sequencing required
• Completely Unknown dna can be sequenced
• only 400bp can be sequenced
• harmful radiolabeled chemicals

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Sanger sequencing:
• first-generation DNA sequencing method
• chain termination method
• dideoxynucleotide sequencing - of the use of the special types of ddNTPs. The
ddNTPs are different from normal dNTPs
• requires prior knowledge of at least 15 - 20 bases of the sample sequence-primer
preparation
• single-stranded DNA template, a DNA primer, a DNA polymerase, normal
deoxynucleotide triphosphates (dNTPs), and modified di-deoxynucleotide
triphosphates (ddNTPs), the latter of which terminate DNA strand elongation
• ddNTPs may be radioactively or fluorescently labelled for detection in automated
sequencing machines

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• The DNA sample is divided into four separate sequencing reactions
• Each contains all four of the standard deoxynucleotides (dATP, dGTP,
dCTP and dTTP) and the DNA polymerase.
• To each reaction only one of the four ddNTPS is added (ddATP, ddGTP,
ddCTP, or ddTTP),
• four separate reactions are needed in this process to test all four
ddNTPs.
• Heat denatured - single stranded

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Components of Manual DNA sequencing

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• After the sequencing reaction is complete the products are run on a
polyacrylamide gel capable of resolving fragments differing by a single
base
• Four lanes are required for each sequence
• The gel plates are removed and the gel is transferred to filter paper
and dried
• The fragments were then visualized by autoradiography
• Finally the sequence was read manually using the eye and a ruler

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Semi automated Sangers
• PAGE method isn’t capable of separating all the fragments in a
single reaction.
• 4 separate tubes and PAGE are required
• Therefore, alternatively, the capillary gel electrophoresis method
can be practiced.

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• Initial steps are same – but here only fluorescent dyes are used
• All 4 labelled ddNTPs are placed in single reaction tube
• At end of the process the fragments are passed through an acrylic gel
capillary tube
• Smaller fragments move faster towards positive charge and hence are
placed at the end of the capillary
• A laser beam is passed to excite the band
• The specific colour of light can be tied to specific nucleotide

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Differeence
• Chemical cleavage
• Radiolabelled
• Base-specific cleavage of DNA by
certain chemicals
• Four different chemicals, one for
each base
• A set of DNA fragments of different
sizes
• DNA fragments contain up to 500
nucleotides
• Chain termination by modified dNTPs
• Fluorescent dye/Radio
• DNA synthesis reactions in four
separate tubes
• The last base in each of these
fragments is known.
• 700 to 1000 base pairs can be
read

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Limitations
• Sanger methods can only sequence short pieces of DNA--about
300 to 1000 base pairs.
• The quality of a Sanger sequence is often not very good in the
first 15 to 40 bases because that is where the primer binds
• Sequence quality degrades after 700 to 900 bases

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NGS
• In principle, the concepts behind Sanger vs. next-generation
sequencing (NGS) technologies are similar.
• In both NGS and Sanger sequencing DNA polymerase adds
fluorescent nucleotides one by one onto a growing DNA
template strand. Each incorporated nucleotide is identified by its
fluorescent tag.
• Critical difference between Sanger sequencing and NGS is
sequencing volume.
• NGS is massively parallel, sequencing millions of fragments
simultaneously per run.

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NGS work flow

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NGS
• Term used to describe several modern sequencing technologies
• Platforms that could provide massively parallel sequencing and
enable millions of DNA fragments to be sequenced
simultaneously
• NGS platforms achieve sequencing in different ways
• Methodology
• Template preparation
• Sample preparation
• DNA fragmentation
• Sequencing and imaging
• Data analysis

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• Nucleic acid sample is fragmented and the ends of the DNA
fragments are ligated with chemically synthesized DNA
molecules of which the nucleotide sequence is already known -
adaptors

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Steps
• Sample preparation
• DNAs/RNAs are extracted from sample like blood or tissue or sputum
• RNA- reverse transcribed to cDNA
• DNA fragmentation
• Random fragmentation of the cDNA or DNA, typically by enzymatic treatment-
reverse transcriptase
• Library preparation
• Small fragments of DNA are tagged with adaptor
• Amplification
• Emulsion PCR
• Bridge PCR
• Rolling circle amplification

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• Sequencing - Sequencing by reversible terminator chemistry
• Fluorescently tagged modified nucleotides + primer
• These labelled nucleotides form base pair with single stranded DNA
• These nucleotides are chemically blocked such that each incorporation is a
unique event, i.e reaction stops after each addition as 3’ end is bound to a
chemical &not free.
• These terminators are similar to ones used in sangers but differ in the way
that they are reversible.
• After each addition a laser beam is passed through the flow cell and each of
the four bases emits its own colour that are recorded by the computer.
• Followed by chemical cleaving of the blocked group and addition of -OH
group preparing the strand for incorporation of the next base pair.
• This process continues till all the bases are read

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• Single-molecule-fluorescence based high-speed DNA sequencing -
Solexa sequencing – Illumina dye sequencing
• Currently regarded as the most widely used platform
• Automated, quick, highly accurate, capable of sequencing multiple
strands simultaneously via massive parallel sequencing, and
economically cheaper in case of whole genome sequencing.
• Shankar Balasubramanian and David Klenerman from Cambridge
University -1998

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Massive parallel sequencing
• Sample multiplexing and pooling
• DNA barcoding

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PROF.S.SUBBIAH et al.
Thank you