The document discusses the history and various methods of DNA sequencing. It begins with early discoveries in DNA sequencing from the 1860s to 1970s. It then describes the basic steps in DNA sequencing including sample preparation, PCR amplification, purification, sequencing preparation, and data analysis. Specific traditional methods like Sanger sequencing and Maxam-Gilbert sequencing are explained in detail. The document also discusses newer next-generation sequencing technologies and how they enable massively parallel sequencing of millions of DNA fragments simultaneously.

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