TECHNICAL REVIEW

TITLE :

DNA SEQUENCINGDNA SEQUENCING

PRESENTED BY:

NOR KHATIJAH MOHD ARIS

DNA sequencing

The power of it is the ability to reduce genes and genomes to chemical entities of defined structure

Objectives :

INTRODUCTION

Objectives :

1) To characterize new clone of cDNA2) To confirm the identity of clone or mutation3) To check fidelity of a newly created

mutation, ligation junction or product of PCR

4) Screening tool to identify polymorphisms and mutation in genes of particular interest

INTRODUCTION

Two of the earliest methods developed for the rapid sequencing of DNA are :

Chemical cleavage method by Allan Maxam and Walter Gilbert.

Chain terminator or dideoxy method by Frederick Sanger.

The Sanger dideoxy method became more popular because of its simplicity and was the chosen method for DNA sequencing.

The sequence of DNA

constitutes the heritable

genetic information in nuclei,

plasmids, mitochondria, and

chloroplasts that forms the

basis for the developmental

programs of all living

organisms.

DNA POLYMERIZATION

• Understand the chemistry of • Understand the chemistry of DNA synthesis

DNA SEQUENCING

“CHAIN TERMINATION METHOD”

NOBEL PRIZE IN CHEMISTRY (1980)

FREDERICK SANGER

1970

MAXAM-GILBERT

1973

ENZYMATIC

SYNTHESIS

CHEMICAL

MODIFICATION

AUTORADIOGRAPHY

‘A technique in which radioactive molecules make their location known by exposing photographic films or emulsions’

Principle of PAGE

• Polyacrylamide gels are chemically cross-linked gels forming by the polymerization of acrylamide with a cross-linking agent, usually N, N’-methylene bisacrylamide (Bis).

• The polymerization initiates by free radical formation usually carrying out with ammonium per sulfate as the initiator and N, N, N’, N’-tetramethylene diamine (TEMED) as a catalyst.

CONVENTIONAL

METHOD…

tetramethylene diamine (TEMED) as a catalyst.

• The length of the chain may be determined by the concentration of acrylamide in the polymerization reaction. Denaturing gels polymerized in the presence of urea agent to suppress base pairing in nucleic acids.

• Denatured DNA migrates through these gels at a rate that is almost completely independent of its base composition and sequence also molecular weight.

MAJOR ADVANTAGE :The sequencing can be performed in asingle reaction

Each of the four ddNTP chain terminators is labeled with a different fluorescent dye,each fluorescing at a different wavelength

AUTOMATED SEQUENCING

each fluorescing at a different wavelength

Utilized fluorescent detection system rather than autoradiogrphy to visualized the product of DNA sequencing reaction

Capillary electrophoresis applied in automated sequencing

DNA sequencing by Capillary Electrophoresis

• ABI 3130xl Genetic Analyzer

– Automated capillary electrophoresis system

– Separate, detect and analyze 16 capillaries of fluorescently labeled DNA fragments in one runrun

• Capillary electrophoresis

– A family of techniques used to separate a variety of compounds

– Driven by electric field performed in narrow tube

– Rapid separation of many hundreds of different compounds

DNA sequencing by Capillary

Electrophoresis

AdvantagesHigh efficiencies, requires minute amounts of sample, automated

Dye molecules were excited by a laser beam and the fluorescent amplified and detected

by photomultiplier tubes

What is the steps

prior sequencing?

PCR

PURIFY PCR PROCUCT

CYCLE SEQUENCING ~2 hours

PURIFY CYCLE SEQUENCING PRODUCT

Refer to UMBI’s Standard Of Procedure…

PURIFY CYCLE SEQUENCING PRODUCT

~2 hours

DENATURE ~2 minutes

DNA SEQUENCING ~2 hour

ELECTROPHOROGRAM

Cycle

sequencing

• Or linear amplification to generate a single

stranded template for sequencing by the

Sanger method

• The key to consistent success in cycle

sequencing is the cleanliness of the template

DNA.

– Impurities of DNA cause premature termination and pausing of DNA polymerase. Whereas oligonucleotides produced by degradation of DNA or RNA cause false priming

Cycle sequencing reactions

The reactions are as below :

- BigDye Terminator V3.1

- Sequencing buffer

- Primer (rev or fwd)

- Template - Template

- distilled water

Three steps in cycle sequencing are

denaturation, annealing and extension

(please refer to UMBI’s SOP)

Gel

electrophoresis

Auto-

sequencer

T-RedT-Red

A-Green

G-Black

C-Blue

ELECTROPHOROGRAMThe result of sequencing

AmpliTaq

DNA polymerase, Fs

rTth pyrophosphatase

EDTA

Sodium Acetate

Purification of

cycle sequencing(DNA precipitation)

BigDye Terminator V3.1 kit(Cycle sequencing or linear amplification)

Refer to UMBI’s Standard Of Procedure…

Dye terminators

Deoxynucleoside

Triphosphates

rTth pyrophosphatase

Magnesium chloride

Hi Di Formamide

Absolute EtOH

Denature (ds ~ ss)

• AmpliTaq DNA polymerase, Fs- Used for fluorescence-based sequencing

- Mutant form of Taq DNA polymerase contains point mutations that eliminates exonuclease activity and reduce discrimination for dNTP over ddNTP which leads to much more even peak intensity pattern

BigDye Terminator V3.1 kit

(Cycle sequencing or linear amplification)

intensity pattern

• rTth pyrophosphatase- Thermally stable inorganic pyrophosphatase that cleaves the

inorganic pyrophosphatase (PP) byproduct of the extension reaction and prevents its accumulation in the sequencing reaction. High [PP] could cause reverse polymerization reaction in sequencing

- Used to prevent reduction in band intensity during prolonged incubation of sequencing reactions

• MgCl2- A necessary cofactor for DNA polymerase activity

- Cofactor - An organic molecule or ion (usually a metal

ion) that is required by an enzyme for its activity.

• dNTP

Continue…

• dNTP

- the building blocks from which the DNA polymerase

synthesizes a new DNA strand.

• Dye terminators

– dRhodamine terminators. Each of the 4 ddNTPs is tagged with different fluorescent dye

• After the sequencing reaction is completed, the reaction

product is cleaned using ethanol precipitation to remove

excess fluorescently labelled terminators.

Purification of cycle sequencing(DNA precipitation)

• The addition of sodium acetate and EDTA enables more

complete removal of terminators leading to cleaner

sequence at the start of the sequence.

Purification of cycle sequencing

(DNA precipitation)

• EDTA– Is a chelating agent and it reduces effective concentration of free

magnesium ions which are necessary for full activity for DNA polymerase

• Sodium acetate • Sodium acetate – To neutralize the charge on the sugar-phosphate backbone of the

DNA. For precipitation, ion pairs between polyanion (DNA) and the cation (sodium) are needed

• Absolute ethanol– Decrease the dielectric constant (e) of the solution. As e goes down,

electrostatic force (F) goes up finally anion and cation pair and result in precipitation DNA based on Coulomb’s Law

F = (Q1*Q2) ÷ (e*r2)

• Hi Di Formamide

- Denature DNA strands

- Resuspend the DNA samples before starting a sample run

- Resuspend calibration standards during the preparation of a calibration or sample run

Denaturing dsDNA

calibration or sample run

- Maintain the electrical connection between the polymer in the capillaries and the injection wells in the electrophoresis chamber by acting as an electrolyte

- To produce an accurate sequence reading

- Do not left the samples too long before running. On exposure to air, Hi Di Formamide breaks down into formic acid. Formic acid can cause anomalous G peaks in the sequence which mask the

true sequence.

• POP-7™

- Performance Optimized Polymer

- ABI 3130xl analyzers use the POP-7™ polymer to

separate long DNA fragments in short run times.

- Replaceable sieving media that separates the DNA

fragments by size during electrophoresisfragments by size during electrophoresis

- Stable in instrument for 7 days. Store at 4°c

• Capillary array

- 16 capillaries – silica capillaries that when filled with polymer enable the separation of the fluorescently labeled DNA fragments by electrophoresis

Shotgun Sequencing Analysis

– A method for determining the sequence for a very large piece of DNA. The basic DNA sequencing reaction can only get the sequence of a few hundred nucleotides.

– For larger ones (like BAC DNA), the DNA were fragmented and inserted the resultant pieces into a convenient vector (a plasmid) to replicate them. After sequenced the fragments, we try to deduce from them the sequence of the original BAC DNA

– The goal is to create a library of overlapping clones that provide – The goal is to create a library of overlapping clones that provide at least fivefold sequence redundancy

over the entire target fragment

NEXT GENERATION SEQUENCING

TECHNOLOGY ?

• Genome Sequencer FLX System

ROCHE/454 [FLX]

- Pyrosequencing or emulsion based PCR

• Applied Biosystems [SOLID] • Applied Biosystems [SOLID]

- Sequencing by Oligonucleotide Ligation

and Detection

• SOLEXA Sequencing Technology

(ILLUMINA)

- Sequencing by Synthesis

References

1. Molecular Cloning ; A Laboratory

Manual by Joseph Sambrook and

David W. Russell. Cold Spring

Harbor Laboratory.

2. Molecular Biology of The Gene by 2. Molecular Biology of The Gene by

Robert F. Weaver. Mc Graw Hill

ENZYMATIC SYNTHESIS

KEY PRINCIPLE :The use of dideoxynucleotides triphosphates (ddNTPs)

as DNA chain terminators

ddNTP : lacking a 3’-0H group required for the formation

of phosphodiester bond between 2 nucleotides during

DNA strand elongation

REQUIRES : - ssDNA as template (isolated from recombinant M13 bacteriophage)

- Bacteriophage T7 DNA polymerase (Sequenase)

- Radioactively or fluorescently labeled nucleotides

- Modified nucleotides that terminates DNA strand elongation (ddNTP)

More efficient and rapidly became the method of

choice because it uses fewer toxic chemicals and

lower amounts of radioactivity

These analogs lack the 3’- OH group required to form the next phosphodiester bond with the incoming nucleotide.

DNA replication terminates at the site where a dideoxy analog (ddNTP) is incorporated.

P

O

HO O

H

P

O

OH

HO

O

O

CH2

NH2

N

N

N

N

O

O

NHN

NH

N

N

2’3’2’3’dideoxydideoxy--

nucleotidesnucleotidesTerminateTerminate

H OH

P

O

OH

O

O

CH2

OH

H

O

H

H

OCH

2

HOH

P

O

O

HO

O

O

CH2

NH2NN

N O

NH2

N

TerminateTerminateDNADNA

ReplicatonReplicaton OH

P

O

HO

O

O

CH2

HO

H

P HO

O

O

CH2

O

H2O

“The Sanger Method”CONVENTIONAL METHOD…

• End-label a DNA fragment we want to sequence (5’ or 3’ labeling)

• Modify one kind of base eg. dimethyl sulfate (DMS) to methylate guanines under mild conditions

MAXAM – GILBERTCHEMICAL MODIFICATION METHOD

• Next, use a reagent (piperidine) that does two things ; causes loss of the methylated base, then it breaks the DNA backbone at the site of the lost base.

• In this case, G in the middle in the sequence was methylated, so strand breakage occurred there

• Finally electrophorese the products and detect by autoradiography

Base Specific modification

G Methylation of Guanine base by DMS treatment

A + G Piperidine weakens the glycosidic bonds of A and G

causing depurination and strand breakage

CHEMICAL MODIFICATION USED IN MAXAM – GILBERT METHOD

A + Gcausing depurination and strand breakage

C + T Hydrazine opens pyrimidine rings that is susceptible of

removal

C In the presence of 1.5M NaCl, hydrazines specific for C

base only

A > C 1.2N NaOH at 90°c results in strong cleavage at A

residues and weaker cleavage at C residues.

5’ 32P-GATCCAAGT 3’

90°C piperidine (1M in H20) is used to cleave sugar phosphate

G

Methylation

with DMS

G + A

Piperidine

T + C

Hydrazine

C

1.5M of NaCl only

C reacts to hydrazine

A > C

1.2N NaOH

chain of DNA at the site of chemical modification

G G+A T+C C A>C 3’TGAACCTAG5’