mdfund_Unit12DNAsequencing

7/28/2019 mdfund_Unit12DNAsequencing

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Chapter 10

DNA Sequencing


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Objectives

Compare and contrast the chemical (Maxam/Gilbert)

and chain termination (Sanger) sequencing methods.

List the components and molecular reactions that occur

in chain termination sequencing. Discuss the advantages of dye primer and dye

terminator sequencing.

Derive a text DNA sequence from raw sequencing data.

Describe examples of alternative sequencing methods,such as bisulfite sequencing and pyrosequencing.


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Sequencing Methods

Maxam/Gilbert chemical sequencing

Sanger chain termination sequencing

Pyrosequencing

Array sequencing


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Maxam-Gilbert sequencing is performed by

chain breakage at specific nucleotides.

DMS

G

GG

G

FA

GA

GG

AG

AA

H

CT

TC

TC

CT

H+S

CC

C

C

Maxam-Gilbert Sequencing


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Sequencing gels are read from bottom to top (5to 3).

G G+A T+C C

3

A

A

G

CA

A

C

G

T

G

CA

G

5

Longer fragments

Shortest fragmentsG

A

Maxam-Gilbert Sequencing


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Chain Termination (Sanger)

Sequencing

A modified DNA

replication reaction.

Growing chains areterminated by

dideoxynucleotides.


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Chain terminates

at ddG


Sequencing

The 3-OH group necessary for formation

of the phosphodiester bond is missing in

ddNTPs.


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Template area to be sequenced

-3OHTCGACGGGC

5OP-

Primer

Template


Sequencing

A sequencing reaction mix includes labeledprimer and template.

Dideoxynucleotides are added separately toeach of the four tubes.


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ddATP + ddAfour dNTPs dAdGdCdTdGdCdCdCdG

ddCTP + dAdGddCfour dNTPs dAdGdCdTdGddC

dAdGdCdTdGdCddCdAdGdCdTdGdCdCddC

ddGTP + dAddG

four dNTPs dAdGdCdTddGdAdGdCdTdGdCdCdCddG

ddTTP + dAdGdCddTfour dNTPs dAdGdCdTdGdCdCdCdG

A

C

G

T


Sequencing


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Sequencing

With addition of enzyme (DNA polymerase), theprimer is extended until a ddNTP isencountered.

The chain will end with the incorporation of theddNTP.

With the proper dNTP:ddNTP ratio, the chainwill terminate throughout the length of the

template.All terminated chains will end in the ddNTP

added to that reaction.


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Sequencing

The collection of fragments is a

sequencing ladder.

The resulting terminated chains areresolved by electrophoresis.

Fragments from each of the four tubes are

placed in four separate gel lanes.


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Sequencing gels are read from bottom to top (5to 3).

G A T C

3

G

G

T

A

AA

T

C

A

T

G5

Longer fragments

Shorter fragmentsddG

ddG


Sequencing


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Cycle Sequencing

Cycle sequencing is chain termination

sequencing performed in a thermal cycler.

Cycle sequencing requires a heat-stable

DNA polymerase.


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Fluorescent Dyes

Fluorescent dyes are multicyclic

molecules that absorb and emit

fluorescent light at specific wavelengths. Examples are fluorescein and rhodamine

derivatives.

For sequencing applications, thesemolecules can be covalently attached to

nucleotides.


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Fluorescent Dyes

In dye primersequencing, the primer contains

fluorescent dyeconjugated nucleotides,

labeling the sequencing ladder at the 5ends of

the chains.

In dye terminatorsequencing, the fluorescent

dye molecules are covalently attached to thedideoxynucleotides, labeling the sequencing

ladder at the 3ends of the chains.

ddA

ddA


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AC

GT

The fragments aredistinguished by size and

color.

Dye Terminator Sequencing

A distinct dye or color is used for each of the

four ddNTP.

Since the terminating nucleotides can be

distinguished by color, all four reactions can be

performed in a single tube.

A

T

G

T


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Capillary

GTCT

GA

Slab gel

GATCG A T C


The DNA ladder is resolved in one gel

lane or in a capillary.


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The DNA ladder is read on an electropherogram.

CapillarySlab gel

5AGTCTG

Electropherogram



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5AGTCTG 5AG(T/A)CTG 5AGACTG

T/T T/A A/A

Automated Sequencing

Dye primer or dye terminator sequencing on capillary

instruments.

Sequence analysis software provides analyzed

sequence in text and electropherogram form.

Peak patterns reflect mutations or sequence changes.


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Alternative Sequencing Methods:

Pyrosequencing

Pyrosequencing is based on the generation

oflight signal through release of

pyrophosphate (PPi) on nucleotide addition.

DNAn + dNTP DNAn+1 + PPI

PPi is used to generate ATP from adenosine

phosphosulfate (APS).

APS + PPI ATP

ATP and luciferase generate light by

conversion of luciferin to oxyluciferin.


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DNA sequence: A T C A GG CC T

Nucleotide added : A T C A G C T


Pyrosequencing

Each nucleotide is added in turn.

Only one of four will generate a light signal.

The remaining nucleotides are removed

enzymatically. The light signal is recorded on a pyrogram.


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Bisulfite Sequencing

Bisulfite sequencing is used to detect

methylation in DNA.

Bisulfite deaminates cytosine, makinguracil.

Methylated cytosine is not changed by

bisulfite treatment. The bisulfite-treated template is then

sequenced.


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The sequence of treated and untreated

templates is compared.

GTC

Methylated sequence: GTCMeGGCMeGATCTATC MeGTGCA

Treated sequence:Me

GGCMe

GATUTATCMe

GTGUA

DNA Sequence:

(Untreated) reference: ...GTCGGCGATCTATCGTGCA

Treated sequence: ...GTCGGCGATUTATCGTGUA

This sequence indicates that these Cs are methylated.


Bisulfite Sequencing


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Summary

Genetic information is stored in the order orsequence of nucleotides in DNA.

Chain termination sequencing is the standard

method for the determination of nucleotidesequence.

Dideoxy-chain termination sequencing has beenfacilitated by the development of cycle sequencingand the use of fluorescent dye detection.

Alternative methods are used for specialapplications, such as pyrosequencing (forresequencing and polymorphism detection) orbisulfite sequencing (to analyze methylated DNA).

mdfund_Unit12DNAsequencing

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