DNA Sequencing Methods:

Chain termination method

Maxam-Gilbert Method

F. Sanger

2nd

generation sequence methods

Pyrosequencing

454 technology

Bridge PCR

Illumina Massively Parallel System

Emulsion PCR

SOLiD system sequencing

3rd

generation sequencing method

Single Molecule Sequencing

The Maxam-Gilbert Method

One of the very first methods invented.

Extremely popular for some time

Could use purified DNA directly instead of clones

Uses chemical cleavage and separation of fragments

Procedure

Purified dsDNA is denatured into ssDNA

ssDNA labeled with 32-P

Sample is separated into 4 reaction groups, one for each dNTP

G+A

o Stock treated with a limited amount of DMS (dimethyl sulfate)

+ formic acid under alkaline conditions to attach a methyl

group to the purine ring:

Leads to instability, releases the ring

o Piperidine facilitates β-elimination, DNA cleaved into 5’ and 3’

fragments

G

o Stock treated with a limited amount of DMS (dimethyl sulfate)

under alkaline conditions to attach a methyl group to the purine

ring followed by Piperidine

C + T

o Stock treated with Hydrazine, which attacks at 4C & 6C and

opens the ring

o Pyridine causes β-elimination and separation into 5’ and 3’

fragments.

C o Stock treated with Hydrazine in 2M NaCl, which attacks at 4C

& 6C and opens the ring followed by Pyridine

The products of cleavage are then analyzed by electrophoresis on a

polyacrylamide gel

Gels are refrigerated with x-ray film, exposed by the radiation from the

isotopes

Films are read from bottom to top

Base calling involve interpreting the banding pattern relative to four chemical

reactions.

Eg a band in C & in CT is read as C, where as a band in CT only & not in C

is read as T.

Similarly for G & GA band

Drawbacks Of Maxam-Gilbert Method

Not really used anymore

Reagents difficult to pack in kits

A large amt of radioactive substance(S35

,P32

) used

Hydrazine used happen to be neurotoxin

Scale-up is difficult

Better methods available

Sanger method

Also called the “dideoxynucleotide” method.

Utilizes dideoxynucleotides, nucleotide bases with no hydroxyl group on 2’ or 3’ C.

Components needed are

DNA template which is needed to be sequenced

A short DNA Primer complementary to the template DNA

DNA polymerase

Four deoxynucleotides (dNTPS)

Four radio-labeled dideoxynucleotides (ddNTPS)

Steps of reactions

Reaction mixture: Four reactions tubes are taken and mix all the components

in all the tubes, with each ddNTPs in separate tube (i.e ddGTPs, ddATPs,

ddCTPs and ddTTPs in respective tubes)

Denaturation: To start the sequencing reaction the mixture is heated so the

complementary template DNA strand separates

Annealing: The temperature is lowered to allow the primer sequence to bind

to its complementary sequence in the template DNA

Extension: Temperature is then slightly raised so that the enzyme polymerase

3 combines to DNA and creates the new strand of DNA

Chain termination:

dNTPs are added by the enzyme until a ddNTP is added. Once a

ddNTP is incorporated into the strand, the chain is terminated.

The strand can be terminated at any position resulting in a collection of

DNA strands of many different lengths.

This results in four dideoxystrands in their respective tubes

Electrophoresis: Next, each reaction mixture is electrophoresed in a separate

lane (4 lanes) at high voltage on a polyacrylamide gel.

Pattern of bands in each of the four lanes is visualized on X-ray film.

Location of “bands” in each of the four lanes indicates the size of the fragment

terminating with a respective radio-labeled ddNTP.

DNA sequence is deduced from the pattern of bands in the 4 lanes

Automation:

Automated sequencers use 4 different fluorescent dyes as tags attached to the

dideoxy nucleotides and run all 4 reactions in the same lane of the gel.

Chromatograms processed with software to sort signals, results in dsDNA

sequence

Two types of machines used for this

Capillary sequencer

Gel-based sequencer

Pyrosequencing

It is a technique to sequence DNA by using chemiluminescent enzymatic reactions

Principle:

First step is the preparation of single stranded DNA molecule as a starting

material by denaturation

DNA polymerase will start elongation by using dNTPs

If the dNTP is incorporated it will release phosphate

(DNA)n + dNTP Polymerase (DNA)n+1 + PPi

Pyrophosphate will be converted into ATP from Adenosine phosphosulphate

(APS) by sulfurylase

APS + PPi Sulfurylase ATP

Luciferase will use ATP to oxidise luciferin and generate a flash of

chemiluminescence

Luciferin + ATP Luciferase Oxyluciferin + Light

The light will be generated as a peak for each one type of nucleotides

incorporated

Each peak represents a nucleotide so by this whole sequence can be

determined

454 Technology:

It is based on pyrosequecing

DNA is sheared into 300-800 bp fragments, and the ends are “polished” by

removing any unpaired bases at the ends

Adapters are added to each end to hold primer. The DNA is made single

stranded at this point

One adapter contains biotin, which binds to a streptavidin-coated bead. The

ratio of beads to DNA molecules is controlled so that most beads get only a

single DNA attached to them

Oil is added to the beads and an emulsion is created. PCR is then

performed, with each aqueous droplet forming its own micro-reactor.

Each bead ends up coated with about a million identical copies of the original

DNA.

After the emulsion PCR has been performed, the oil is removed, and the beads

are put into a “picotiter” plate. Each well is just big enough to hold a single

bead

Reagents are then added to the beads places in microwells :

o DNA polymerase

o Adenosine Phosphosulfate (APS)

o ATP Sulfurylase

o Luciferin

o Luciferase

The plate is then repeatedly washed with the each of the four dNTPs in a

repeating cycle

The plate is coupled to a fiber optic chip. A CCD camera records the light

flashes from each well

Illumina Massively Parallel System

Principle

o The idea is to put 2 different adapters on each end of the DNA, then bind it

to a slide coated with primers complementary sequences for each adapter.

o This allows “bridge PCR”, producing a small spot of amplified DNA on

the slide

o The slide contains millions of individual DNA clusters.

o The spots are visualized during the sequencing run, using the fluorescence

of the nucleotide being added

Steps involved

o Prepare genomic DNA sample

Randomly fragment genomic DNA and ligate adapters to both end

of the fragments

o Attach DNA to surface

Bind ss fragments randomly to the inside surface of the flow cell

channels (just like a sheet)

o Bridge amplification

Add unlabeled nucleotides and enzymes to initiate the solid phase

bridge amplification

o Fragment become double stranded

The enzyme incorporate the nucleotide to form double stranded

DNA on a solid phase substrate

o Denature the double stranded molecules

Denaturation leaves single stranded substrate anchored to the solid

phase substrate (basically it is surface attached)

o Complete amplification

After multiple cycles amplification completes

o Determine first base

Add four labelled reversible terminators, primers and DNA

polymerase to the flow cell

o Image first base

After laser excitation, capture the image of emitted fluorescence

from each cluster of flow cell

o Determine second base

Remove the terminal label and add all four reversible terminator

bases

o Image second base

Laser excitation, record the second base

o Repeat the cycle until 33-36 bases read.

o Align data, compare to the reference, identify sequence

Single molecule sequencing

o Single molecule real time sequencing (also known as SMRT) is a parallelized single

molecule DNA sequencing by synthesis technology.

o Relatively new, “Next-Next-Gen” technique that does not require the use of cloning,

amplification, or ligation

o Sequence-by-Synthesis approach

o May significantly reduce the cost of sequencing

o Applications in:

Pharmaceutical R&D

Oncology Research

Clinical Diagnosis/Personalized Medicine

o It requires Zero Mode Waveguide (ZMW)

Is a nano-photonic, cylindrical metallic visualization

chamber, about 70nm wide

This is present in SMRT 8 cell pacs (a tray of 8

packs, a total of 12 SMRT 8 cells pacs can be

loaded)

Providing a detection volume equivalent to 1 base.

Creates an illuminated observation that is small

enough to observe only a single nucleotide of DNA

being incorporated

o Steps

A DNA polymerase molecule is affixed to the bottom of a Zero Mode

Waveguide with a single molecule of DNA

Phospholinked nucleotides tagged with different colored fluorophores are

introduced in high concentrations

DNA polymerase incorporates a nucleotide (this takes place within the

detection volume for a very short period of time)

The engaged fluorophore emits light with color corresponding to base

identity

When DNA polymerase cleaves the bond holding the fluorophore, the dye

diffuses out and the signal returns to baseline

o Advantages over older methods:

Long read length

Short cycling time

Low cost

Reliably good data

Rate of reaction within the detection volume is very fast, results in low

background

Low detection volume (20 zeptoliters, or 20x10^-21 Liters) decreases

chance of interference