DNA Sequencing

DNA sequencingDetermination of nucleotide sequence

the determination of the precise sequence of nucleotides in a sample of DNA

Two similar methods:1. Maxam and Gilbert method

2. Sanger method

They depend on the production of a mixture of oligonucleotides labeled either radioactively or fluorescein, with one common end and differing in

length by a single nucleotide at the other end This mixture of oligonucleotides is separated by high resolution electrophoresis on polyacrilamide gels and the position of the bands

determined

Maxam-Gilbert

• Walter Gilbert– Harvard physicist– Knew James Watson– Became intrigued with

the biological side– Became a biophysicist

• Allan Maxam

The Maxam-Gilbert Technique

• Principle - Chemical Degradation of Purines– Purines (A, G) damaged by

dimethylsulfate– Methylation of base– Heat releases base– Alkali cleaves G– Dilute acid cleave A>G

Maxam-Gilbert Technique

• PrincipleChemical Degradation of Pyrimidines– Pyrimidines (C, T) are

damaged by hydrazine– Piperidine cleaves the

backbone– 2 M NaCl inhibits the

reaction with T

Maxam and Gilbert MethodChemical degradation of purified fragments (chemical degradation)

The single stranded DNA fragment to be sequenced is end-labeled by treatment with alkaline phosphatase to remove the 5’phosphate

It is then followed by reaction with P-labeled ATP in the presence of polynucleotide kinase, which attaches P labeled to the 5’terminal

The labeled DNA fragment is then divided into four aliquots, each of which is treated with a reagent which modifies a specific base

1. Aliquot A + dimethyl sulphate, which methylates guanine residue2. Aliquot B + formic acid, which modifies adenine and guanine residues3. Aliquot C + Hydrazine, which modifies thymine + cytosine residues4. Aliquot D + Hydrazine + 5 mol/l NaCl, which makes the reaction specific for cytosineThe four are incubated with piperidine which cleaves the sugar phosphate

backbone of DNA next to the residue that has been modified

Maxam-Gilbert sequencing - modifications

Maxam-Gilbert sequencing - summary

Advantages/disadvantagesMaxam-Gilbert sequencing

Requires lots of purified DNA, and many intermediate purification steps Relatively short readings

Automation not available (sequencers) Remaining use for ‘footprinting’ (partial protection against DNA modification when proteins bind to specific regions, and that produce

‘holes’ in the sequence ladder)

In contrast, the Sanger sequencing methodology requires little if any DNA purification, no restriction digests, and no labeling of

the DNA sequencing template

Sanger MethodFred Sanger, 1958

– Was originally a protein chemist

– Made his first mark in sequencing proteins

– Made his second mark in sequencing RNA

1980 dideoxy sequencing

Original Sanger Method

Random incorporation of a dideoxynucleoside triphosphate into a growing strand of DNA

Requires DNA polymerase IRequires a cloning vector with initial primer (M13, high

yield bacteriophage, modified by adding: beta-galactosidase screening, polylinker)

Uses 32P-deoxynucleoside triphosphates

Sanger Method

in-vitro DNA synthesis using ‘terminators’, use of dideoxi- nucleotides that do not permit chain elongation after their

integration DNA synthesis using deoxy- and dideoxynucleotides that

results in termination of synthesis at specific nucleotidesRequires a primer, DNA polymerase, a template, a mixture of

nucleotides, and detection system Incorporation of di-deoxynucleotides into growing strand

terminates synthesisSynthesized strand sizes are determined for each di-deoxynucleotide by using gel or capillary electrophoresis

Enzymatic methods

Dideoxynucleotide

no hydroxyl group at 3’ endprevents strand extension

CH2O

OPPP5’

3’

BASE

The principles• Partial copies of DNA fragments made with DNA

polymerase• Collection of DNA fragments that terminate with

A,C,G or T using ddNTP• Separate by gel electrophoresis

• Read DNA sequence

CCGTAC3’ 5’5’ 3’primer

dNTP

ddATP

GGCA

ddTTP

GGCAT

ddCTP

GGC G

ddGTP

GGGGCATG

A T C G

DideoxyChain Terminator

TemplatePrimer

Extension Chemistry– polymerase– termination

– labelingSeparationDetection

Chain Terminator Basics

TargetTemplate-Primer

ExtendddA

ddG

ddC

ddTLabeled Terminators

ddA

AddC

ACddG

ACG ddT

TGCA

dN : ddN100 : 1

Electrophoresis

Sanger Method Sequencing Gel

Template

ssDNA vectors– M13– pUC

PCRdsDNA (+/- PCR)

Primers

Universal primers– cheap, reliable, easy, fast, parallelparallel

– BULK sequencing

Custom primers– expensive, slow, one-at-a-time

– ADAPTABLEADAPTABLE

Extension

Polymerase– Sequenase

– Thermostable (Cycle Sequencing)

Terminators– Dye labels (“Big Dye”)

• spectrally different, high fluorescence– ddA,C,G,T with primer labels

Separation

Gel ElectrophoresisCapillary Electrophoresis

– suited to automation• rapid (2 hrs vs 12 hrs)• re-usable• simple temperature control• 96 well format

Sample Output

1 lane

Sequencing of DNA by the Sanger method

Comparison

• Sanger Method– Enzymatic– Requires DNA synthesis– Termination of chain

elongation

• Maxam Gilbert Method– Chemical– Requires DNA– Requires long stretches of

DNA– Breaks DNA at different

nucleotides