©2001 timothy g. standish matthew 13:17 17for verily i say unto you, that many prophets and...

©2001 Timothy G. Standish

Matthew 13:17

17 For verily I say unto you, That many prophets and righteous men have desired to see those things which ye see, and have not seen them; and to hear those things which ye hear, and have not heard them.


DNA SequencingDNA Sequencing

Timothy G. Standish, Ph. D.


Sequenced GenomesSequenced Genomes Over the past three years large scale sequencing of

eukaryotic genomes has become a reality Currently the sequencing of at least 5 multi-celled

eukaryotic genomes has been completed: 1998 Caenorhabditis elegans - 8 x 107 bp - A nematode

worm 2000 Homo sapiens - 3 x 109 bp - Humans 2000 Arabidopsis thaliana - 1.15 x 108 - A plant related to

mustard 2000 Drosophila melanogaster - 1.65 x 108 bp - Fruit flies 2002 Anopheles gambiae – 2.78 x 108 bp mosquito vector

of malaria


New TechnologyNew Technology Rapid sequencing of large complex genomes

has been made possible by: Foundational work done over many years

and… Dramatic improvement in DNA sequencing

technology over the past few years In this presentation we will look at both the

basic principles of DNA sequencing and how techniques have been refined to yield the dramatic results we now see


A Sequencing TimelineA Sequencing Timeline1977 Sanger and Maxam-Gilbert

sequencing techniques developed

1980 M13 vector developed for cloning, many refinements and application of computer technology

1990 Improved sequencing enzymes, fluorescent dyes developed, robotics used for high throughput

1997 Sacromycetes Cerevisiae genome sequenced

1999 Caenorhabdits elegans Human chromosome 22 and about 20 bacterial genomes

2000 Drosophila melanogaster, Homo sapiens, Arabidopsis thaliana

= 2,000 bp20 X 100 bp

Total/weekSamples/person/week Average read length

= 18,000 bp60 X 300 bp

= 90,000 bp180 X 500 bp

= 325,000 bp500 X 650 bp

=3,000,000bp5000X 600 bp

= 200 bp4 X 50 bp


Basic PrinciplesBasic Principles All current practical DNA sequencing techniques

can be divided into four major steps:

1. Labeling of DNA so that small quantities can be easily detected, traditionally done by labeling with either P32 or S35

2. Generation of fragments for which the specific bases at the 3’ end are known

3. Separation of fragments using gel electrophoresis sensitive ennough to resolve differenced in size of one nucleotide

4. Fragment detection


OutlineOutline In this presentation we will look at:

1. The Maxam-Gilbert and Sanger methods of DNA fragment generation

2. Then methods for separation of fragments

3. And finally examine how these techniques have been refined and automated to allow for rapid cheap sequencing of large quantities of DNA


The Maxam-GilbertThe Maxam-GilbertChemical MethodChemical Method

Three major steps:

1. DNA to be sequenced is typically labeled at the 5’ end using P32

2. Fragments are generated using chemicals that break DNA at specific bases

3. These fragments are then separated and detected using autoradiography

Polyacylamide Gel Electrophoresis is typically used to separate fragments on the basis of single nucleotide differences


2 Fragment Generation2 Fragment GenerationA number of chemicals will specifically

modify the bases in DNAModified bases can then be removed

from the deoxyribose sugar to which they are attached on the sugar-phosphate DNA backbone

Piperidine, a volatile secondary amine, is used to cleave the sugar-phosphate back bone of DNA at sites where bases were modified


Cleavage at Specific BasesCleavage at Specific Bases Typically 5 reactions are run:

1. Dimethylsulfate at pH 8.0 results in modification of guanine (G)

2. Piperidine formate at pH 2.0 breaks glycosidic bonds between deoxyribose and both purines, guanine (G) and adenine (A), by protination of nitrogen atoms

3. Hydrazine (rocket fuel!) opens pyrimidine rings on both pyrimidines, cytosine (C) and thymine (T)

4. Hydrazine in the presence of 1.5 M NaCl only reacts with C

5. 1.2 N NaOH at 90 oC strongly cleaves at A and may also weakly cleave at C


Cleavage at Specific BasesCleavage at Specific Bases The trick in chemical sequencing is to not allow

the reactions to go to completion Partial reactions run using the following

conditions will result in a series of labeled DNA fragments whose final base is known:

Dimethylsulfate at pH 8.0 -----------> G

Piperidine formate at pH 2.0 -------> G and A

Hydrazine ------------------------------> C and T

Hydrazine in 1.5 M NaCl -----------> C

1.2 N NaOH at 90 oC -----------------> A and some C


Partial Reactions:Partial Reactions:Dimethylsulphate pH 8.0Dimethylsulphate pH 8.0P32

5’*NNGACGTACTTA3’








Partial Reactions:Partial Reactions:Dimethylsulphate pH 8.0Dimethylsulphate pH 8.0








Modification of some, but not all, of the G bases as the reaction is not allowed to go to completion


Partial Reactions:Partial Reactions:Dimethylsulphate pH 8.0Dimethylsulphate pH 8.0

5’TACTTA3’

5’ACGTACTTA3’

5’TACTTA3’

5’ACGTACTTA3’

5’ACGTACTTA3’

5’TACTTA3’

5’*NNGAC3’

5’*NN3’

5’*NN3’

5’*NN3’

5’*NN3’

5’*NNGAC3’

Following breaking of the DNA strand at positions where G was chemically modified, two sets of fragments result: 1) A labeled set all ending where a G once was and 2) An unlabeled set which cannot be detected using autoradiography

Unlabeled fragments undetectable using autoradiography

Labeled fragments all of which represent a place where G used to be


Partial Reactions:Partial Reactions:HydrazineHydrazine








Some, but not all, C and T bases are modified as the reaction is not allowed to go to completion


Partial Reactions:Partial Reactions:HydrazineHydrazine

5’*A3’

5’GTACTTA3’

5’*A3’

5’*TA3’

5’G3’ 5’ACTTA3’

5’ACTTA3’

5’*NNGACGTAC3’

5’*NNGA3’

5’*NNGACGTACT3’

5’*NNGACGTAC3’

5’*NNGA3’

5’*NNGACG3’

Following breaking of the DNA strand at positions where C or T was chemically modified, two sets of fragments result: 1) A labeled set all ending where a C or T once was and 2) An unlabeled set which cannot be detected using autoradiography

Unlabeled fragments

LabeledT C set


DisadvantagesDisadvantagesToxic chemicalsLarge amounts of radioactivitySometimes ambiguous and

frequently ugly sequencing gelsTricky to read autoradsLack of automated methods


Sanger SequencingSanger Sequencing The Sanger sequencing method takes advantage of

the way that normal DNA replication occurs For DNA to be extended using normal DNA

polymerases, a hydroxyl group must be present at the 3’ carbon on deoxyribose

Fragments are generated by spiking reactions with small quantities 2’ 3’ dideoxy nucleotides which terminate polymerization whenever they are incorporated into DNA

Polymerases used must lack 3’ to 5’ exonuclease proof reading activity for this method to work


H

P

O

OH

OH

HO

O

O

CH2

NH2

N

N N

N

Sugar

Base

Phosphate

3’

5’

2’

1’4’

DideoxynucleotidesDideoxynucleotides DNA Sequencing using the Sanger

method involves the use of 2’3’-dideoxynucleotide triphosphates in addition to regular 2’-deoxynucleotide triphosphates

Because 2’3’-dideoxynucleotide triphosphates lack a 3’ hydroxyl group, and DNA polymerization occurs only in the 3’ direction, once 2’3’-dideoxynucleotide triphosphates are incorporated, primer extension stops

H

2’3’-dideoxynucleotide monophosphate

2’-dideoxynucleotide monophosphate

SU

GA

R-P

HO

SP

HA

TE

BA

CK

BO

NE

H

P

O

HO

O

O

CH2

HOH

P

O

O

HO

O

O

CH2

H

P

O

OH

HO

O

O

CH2

NH2

N

N

N

N

O

O

NH2N

NH

N

N

N O

NH2

N

B A

S E

S

2’3’2’3’dideoxy-dideoxy-

nucleotidesnucleotidesTerminateTerminate

DNADNAReplicatonReplicaton OH

P

O

HO

O

O

CH2

HO

O

H 2N

NHN N

N H

H OH

P

O

OH

O

O

CH2

CH 3

O

O

HNN

OH

H

P HO

O

O

CH2

HO

N

O

H 2N

N

H2O

2’3’did

eoxynu

cleotide


Making DNA FragmentsMaking DNA Fragments In Sanger DNA sequencing reactions all the basic

components needed to replicate DNA are used 4 reactions are set up, each containing:

– DNA Polymerase

– Primer

– Template to be sequenced

– dNTPs

– A small amount of one ddNTP ddATP, ddCTP, ddGTP, ddTTP

As incorporation of ddNTPs terminates DNA replication, a series of fragments is produced all terminating with the ddNTP that was added to each reaction


DNA SequencingDNA Sequencing

Plasmid (or phage) with cloned DNA

fragment

Primer Binding sites

Cloned fragment

Primer


The ddATP ReactionThe ddATP Reaction

5’TTATCG3’AATAGCATGGTACTGATCTTACGCTAT5’

5’TTATCGTACCATGACTAGATGCGA

5’TTATCGTACCA

5’TTATCGTACCATGACTA

5’TTATCGTA

5’TTATCGTACCATGA

5’TTATCGTACCATGACTAGATGCGATA

5’TTATCGTACCATGACTAGA

Pol.5’TTATCGTA Let me

Through!

Pol.5’TTATCGTACCATGA

Oh comeon!

Pol.5’TTATCGTACCATGACTAGA

NotAgain!

Pol.5’TTATCGTACCATGACTAGATGCGATA

Agggg….


Separation of DNA FragmentsSeparation of DNA FragmentsAll current practical sequencing

methods rely on separation of DNA fragments in such a way that differences in length of a single base can be resolved

This is typically done using polyacrylamide gel electrophoresis


Acrylamide

Acrylamide

Polyacrylamide GelsPolyacrylamide Gels Polyacrilamide is a polymer made of acrylamide

(C3H5NO) and bis-acrilamide (N,N’-methylene-bis-acrylamide C7H10N2O2)

O

CH

CH2

NH2C

O

CHCH2

NH2C

CH2

bis-Acrylamide

O

CHCH2

NH2C

Acrylamide


Polyacrylamide GelsPolyacrylamide Gels

O

CHCH2

NH2C

O

CHCH2

NH2C

SO4-.

Acrylamide polymerizes in the presence of free radicals typically supplied by ammonium persulfate


Polyacrylamide GelsPolyacrylamide Gels Acrylamide polymerizes in the presence of free

radicals typically supplied by ammonium persulfate

SO4-.

O

CHCH2

NH2C

O

CHCH2

NH2CNH2

O

CHCH2

C

O

CHCH2

NH2C

TMED (N,N,N’,N’-tetramethylethylenediamine) serves as a catalyst in the reaction


Polyacrylamide GelsPolyacrylamide Gels bis-Acrylamide polymerizes along with acrylamide

forming cross-links between acrylamide chainsO

CHCH2

NH2C

O

CHCH2

NH2C

O

CHCH2

NH2CNH2

O

CHCH2

C

O

CHCH2

NH2C

O

CHCH2

NH2C

bis-Acrylamide

O

CH

CH2

NH2C

O

CHCH2

NH2C

CH2


Polyacrylamide GelsPolyacrylamide Gels bis-Acrylamide polymerizes along with acrylamide

forming cross-links between acrylamide chains


Polyacrylamide GelsPolyacrylamide Gels Pore size in gels can be varied by varying the ratio

of acrylamide to bis-acrylamide

Lots of bis-acrylamideLittle bis-acrylamide

DNA sequencing separations typically use a 19:1 acrylamide to bis ratio


Denaturation of DNADenaturation of DNA For gel electorphoresis to accurately separate on the

basis of size and not shape or other considerations it is important that the DNA be denatured

This is typically achieved by using a high urea concentration (8 M) in the gel

Double stranded DNA

Denatured Single Stranded DNA

8 M8 MUreaUrea

Self annealing DNA

8 M8 MUreaUrea

Denatured Single Stranded DNA


5’GACGTACTTA3’

G G+A T+C C A>CG G+A T+C C A>C

Separation of Fragments:Separation of Fragments:Maxam-GilbertMaxam-Gilbert

1.2 N NaOH at

90 oC

A>C

Hydrazine

T+C

Piperidine formate

pH 2

G+A

Dimethyl sulfate pH 8

G

Hydrazine in 1.5 M

NaCl

CX

XX

XX

X

5’

t

o

3

’


Separation of Sanger FragmentsSeparation of Sanger Fragments Products from 4 reactions

each containing a small amount of a dideoxynucleotide are loaded onto a gel

Because polymerization goes 5’ to 3’ shortest fragments are 5’ compared to longer fragments which are in the 3’ direction

ddTTPddCTP ddGTPddATP

Rea

d 5’

to 3

’ fr

om b

otto

m to

top


To read the autorad it is important to start at the bottom and work up so that it is read in the 5’ to 3’ direction

DNA SequencingDNA SequencingWhat A SequencingWhat A Sequencing

Autorad ActuallyAutorad ActuallyLooks LikeLooks Like

A C G T

5’CTAGAGGATCCCCGGGTACCGAGCT...3’


Sequencing Method RefinementsSequencing Method Refinements Because of difficulties intrinsic to the Maxam-

Gilbert chemical sequencing strategy, efforts at improvement have been concentrated on the Sanger method

Major improvements in the following areas have been achieved

Labeling and detection Fragment separation DNA Polymerases used in sequencing and

resulting strategies for generation of fragments Automation


Pros and Cons of thePros and Cons of theSanger MethodSanger Method

It is more amenable to automation than Maxam-Gilbert

Fewer dangerous chemicals are used, but acrylamide and P32 or S35 are still a problem

Gels or autorads are generally cleaner looking and the reading of bases is a lot easier than Maxam-Gilbert data

The bottom line: Without improvements in automation, detection and separation technologies Sanger sequencing is still very labor intensive


Labeling and DetectionLabeling and Detection Labeling using radioactive isotopes is difficult,

dangerous and expensive Using biotin labeled primers has allowed

conjugation of enzymes to fragments and their subsequent detection using substrates that change color in the presence of the enzyme

This technique is clumsy, expensive, time consuming and unreliable

It also may require transfer of fragments to membranes thus increasing labor and generally has not caught on


Labeling and DetectionLabeling and Detection Another approach has involved development

of very sensitive silver staining technologies I have tried this one, it is miserable and

unreliable Read length on gels is typically short and

creation of a permanent copy of the gel requires expensive additional equipment and supplies

It may not involve isotopes, but it is such a hassle and the data is of such low quality that it is not worth the effort


Labeling and DetectionLabeling and DetectionThe most significant advance in labeling

has been the production of electrophoretically neutral dyes that fluoresce at specific wavelengths when excited by laser produced light over a very narrow range of wavelengths

These dyes, when attached to primers allow detection down to 15 attomoles (10-18)

That’s less than 107 molecules!


The Li-Cor SystemThe Li-Cor System

Fluorescence of dyes attached to DNA fragments are detected as they pass the lasers and detectors

Data in digital form is fed directly into a computer system where automated base calling is done

A graphic representation of the data resembles a traditional autorad with bands appearing in 4 lanes

Li-Cor of Lincoln, Nebraska was one of the first to implement fluorescent dyes as part of an automated sequencing system

The Li-Cor system uses infrared lasers scanning a fixed line toward the bottom of an acrylamide slab gel


Dye labeled fragments

Polyacrylamide gel

The Li-Cor SystemThe Li-Cor SystemA T G C

Detector CCDCCD

ZappoZappo

Laser

…..


Pros and ConsPros and Cons The Li-Cor systems major advantage is the lengths

of its DNA reads– Because all fragments travel through the entire gel,

resolution is sufficient to read over 1,000 bases in a single run with over 99 % accuracy

– This is better than just about any single run manual sequencing method

Elimination of manual reading of autorads also eliminates human error and removes a labor intensive step

P32 or S35 not used - another major advantage Tricky acrylamide gels still must be cast and

loaded manually


Applied BiosystemsApplied Biosystems Applied Biosystems (ABI) has developed fluorescent dye

systems further and improved methods for loading and electrophoresis

Four dyes each of which fluoresce at a different wavelength, but having about the same impact on electrophoritic mobility can be used to label either primers or the nucleotides that terminate a reaction

If terminator dyes are used, the entire sequencing reaction is reduced to one tube from 4 in conventional Sanger sequencing

Instead of polyacrylamide slab gels, a single capillary can be used with a liquid polymer that is replaced after each individual run


3’AATAGCATAACGTTAACGTTACGCTAT5’Pol.

Oh comeon!

5’TTATCGTACCACPol.

5’TTATCGTACCATAATTNot

Again!

Pol.

Agggg….

5’TTATCGTACCATAATTGCA

Replication Using Dye TerminatorsReplication Using Dye Terminators

5’TTATCG

5’TTATCGTATTGCAATTGCA

5’TTATCGTATTGCAATT

5’TTATCGTA

5’TTATCGTATTGCAAT

5’TTATCGTATTGCAATTG

5’TTATCGTATTGCAA

5’TTATCGTATTGCAATTGC

5’TTATCGTATTGCA

5’TTATCGTAT

5’TTATCGTATTG5’TTATCGTATT

5’TTATCGTATTGC

Pol.5’TTATCGTA Let me

Through!

As the base at the end of each fragment is clearly marked with a unique fluorescent dye, the entire reaction can be done in a single tube


…..

Heat plate

Liquid polymer

ATTGC A

ABI Prism 310 SystemABI Prism 310 System

ZappoZappo

Laser

Beam splitter

Detectors

-

+Window

Sequencing reactionSequencing reaction

Capillary


The State of the ArtThe State of the Art The ABI Prism 310 (1 capillary), 3100 (16

capillaries) and 3700 (96 capillaries) represent the current state of the art in automated sequencing machines

A single ABI Prism 377 slab gel sequencer can run 115,000 bases per day!

The 3100 can run up to 184,000 bases per day The 3700 can run up to 1,104,000 bases per day Large sequencing facilities, like Celera, have

factories full of these machines which can run 24 hours a day with very little down time for routine maintenance


The State of the ArtThe State of the Art

ABI Prism 3700

©2001 timothy g. standish matthew 13:17 17for verily i say unto you, that many prophets and...

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