Methods of DNA Sequencing

7/29/2019 Methods of DNA Sequencing

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DNA Sequencing

DNA sequencing used to determine the actualDNA sequence of an organism.


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DNA sequencing

ACGTGACTGAGGACCGTG

CGACTGAGACTGACTGGGT

CTAGCTAGACTACGTTTTA

TATATATATACGTCGTCGT

ACTGATGACTAGATTACAG

ACTGATTTAGATACCTGAC

TGATTTTAAAAAAATATT


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DNA sequencing refers to the methods and technologies

that used to determine the orders of nucleotide bases in

a DNA molecule, namely adenine (A), guanine (G),

cytosine (C) and thymine (T).

The first DNA sequences were obtained in the early

1970s by academic researchers using laboriousmethods based on two-dimensional chromatography.

Following the development of fluorescence-based

sequencing methods with automated analysis, DNAsequencing has become easier and orders of magnitude

faster.

DNA sequencing
http://en.wikipedia.org/wiki/DNA_sequencinghttp://en.wikipedia.org/wiki/Two-dimensional_chromatographyhttp://en.wikipedia.org/wiki/Fluorescencehttp://en.wikipedia.org/wiki/DNA_sequencerhttp://en.wikipedia.org/wiki/DNA_sequencerhttp://en.wikipedia.org/wiki/DNA_sequencerhttp://en.wikipedia.org/wiki/DNA_sequencerhttp://en.wikipedia.org/wiki/Fluorescencehttp://en.wikipedia.org/wiki/Two-dimensional_chromatographyhttp://en.wikipedia.org/wiki/Two-dimensional_chromatographyhttp://en.wikipedia.org/wiki/Two-dimensional_chromatographyhttp://en.wikipedia.org/wiki/Two-dimensional_chromatographyhttp://en.wikipedia.org/wiki/Two-dimensional_chromatographyhttp://en.wikipedia.org/wiki/DNA_sequencinghttp://en.wikipedia.org/wiki/DNA_sequencinghttp://en.wikipedia.org/wiki/DNA_sequencing


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DNA sequencing enables us to perform a thorough

analysis of DNA because it provides us with the most

basic information of all: the sequence of nucleotides.

The knowledge of DNA sequences has formed the basis

of basic biological researches and clinical genetic

diagnosis.

There are also numerous applied technology fields such

as biotechnology, forensic science and biological

systematics that are heavily dependent on theinformation generated through DNA sequencing.

DNA sequencing


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DNA sequencing may be used to determine the

sequence of individual genes, larger genetic regions (i.e.

clusters of genes or operons), full chromosomes or

entire genomes.

Depending on the methods used, sequencing may

provide the order of nucleotides in DNA orRNA isolatedfrom cells of animals, plants, bacteria, archaea, or

virtually any other source of genetic information.

The resulting sequences may be used by researchers inmolecular biology or genetics to further scientific

progress or may be used by medical personnel to make

treatment decisions or aid in genetic counseling.

DNA sequencing
http://en.wikipedia.org/wiki/Genehttp://en.wikipedia.org/wiki/Operonshttp://en.wikipedia.org/wiki/RNAhttp://en.wikipedia.org/wiki/Archaeahttp://en.wikipedia.org/wiki/Molecular_biologyhttp://en.wikipedia.org/wiki/Geneticshttp://en.wikipedia.org/wiki/Genetic_counselinghttp://en.wikipedia.org/wiki/Genetic_counselinghttp://en.wikipedia.org/wiki/Genetic_counselinghttp://en.wikipedia.org/wiki/Genetic_counselinghttp://en.wikipedia.org/wiki/Geneticshttp://en.wikipedia.org/wiki/Molecular_biologyhttp://en.wikipedia.org/wiki/Molecular_biologyhttp://en.wikipedia.org/wiki/Molecular_biologyhttp://en.wikipedia.org/wiki/Archaeahttp://en.wikipedia.org/wiki/RNAhttp://en.wikipedia.org/wiki/Operonshttp://en.wikipedia.org/wiki/Gene


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History

RNA sequencing was one of the earliest forms of nucleotide

sequencing.

The major landmark of RNA sequencing is the sequence of the first

complete gene and the complete genome of Bacteriophage MS2,

identified and published by Walter Fiers in 1972 and 1976. Frederick Sanger developed rapid DNA sequencing methods with

chain-terminating inhibitors" in 1977.

Walter Gilbert and Allan Maxam at Harvard also developed

sequencing methods, including one for "DNA sequencing by

chemical degradation". In 1973, Gilbert and Maxam reported the sequence of 24 basepairs

using a method known as wandering-spot analysis.

Advancements in sequencing were aided by the concurrent

development of recombinant DNA technology, allowing DNA

samples to be isolated from sources other than viruses.
http://en.wikipedia.org/wiki/Bacteriophage_MS2http://en.wikipedia.org/wiki/Walter_Fiershttp://en.wikipedia.org/wiki/Frederick_Sangerhttp://en.wikipedia.org/wiki/Walter_Gilberthttp://en.wikipedia.org/wiki/Allan_Maxamhttp://en.wikipedia.org/wiki/Harvard_Universityhttp://en.wikipedia.org/wiki/Recombinant_DNAhttp://en.wikipedia.org/wiki/Recombinant_DNAhttp://en.wikipedia.org/wiki/Recombinant_DNAhttp://en.wikipedia.org/wiki/Recombinant_DNAhttp://en.wikipedia.org/wiki/Harvard_Universityhttp://en.wikipedia.org/wiki/Allan_Maxamhttp://en.wikipedia.org/wiki/Allan_Maxamhttp://en.wikipedia.org/wiki/Allan_Maxamhttp://en.wikipedia.org/wiki/Walter_Gilberthttp://en.wikipedia.org/wiki/Walter_Gilberthttp://en.wikipedia.org/wiki/Walter_Gilberthttp://en.wikipedia.org/wiki/Frederick_Sangerhttp://en.wikipedia.org/wiki/Frederick_Sangerhttp://en.wikipedia.org/wiki/Frederick_Sangerhttp://en.wikipedia.org/wiki/Walter_Fiershttp://en.wikipedia.org/wiki/Walter_Fiershttp://en.wikipedia.org/wiki/Walter_Fiershttp://en.wikipedia.org/wiki/Bacteriophage_MS2http://en.wikipedia.org/wiki/Bacteriophage_MS2http://en.wikipedia.org/wiki/Bacteriophage_MS2http://en.wikipedia.org/wiki/Bacteriophage_MS2


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The first full DNA genome to be sequenced was that ofbacteriophage X174 in 1977.

Leroy E. Hood's and Smith announced the first semi-automated

DNA sequencing machine in 1986.

In 1995, Venter, Hamilton Smith, and colleagues published the first

complete genome of a free-living organism, the bacteriumHaemophilus influenzae. The circular chromosome contains

1,830,137 bases and its publication in the journal Science marked

the first published use of whole-genome shotgun sequencing,

eliminating the need for initial mapping efforts.

Several new methods for DNA sequencing were developed in themid to late 1990s. These techniques comprise the first of the "next-

generation" sequencing methods.

In 1996, Pl Nyrn and his student Mostafa Ronaghi published their

method ofpyrosequencing.

A year later, Pascal Mayer and Laurent Farinelli describing DNA

History
http://en.wikipedia.org/wiki/Bacteriophage_%CF%86X174http://en.wikipedia.org/wiki/Bacteriophage_%CF%86X174http://en.wikipedia.org/wiki/Bacteriophage_%CF%86X174http://en.wikipedia.org/wiki/Leroy_E._Hoodhttp://en.wikipedia.org/wiki/Hamilton_O._Smithhttp://en.wikipedia.org/wiki/Haemophilus_influenzaehttp://en.wikipedia.org/wiki/P%C3%A5l_Nyr%C3%A9nhttp://en.wikipedia.org/wiki/Mostafa_Ronaghihttp://en.wikipedia.org/wiki/Pyrosequencinghttp://en.wikipedia.org/wiki/Pyrosequencinghttp://en.wikipedia.org/wiki/Mostafa_Ronaghihttp://en.wikipedia.org/wiki/Mostafa_Ronaghihttp://en.wikipedia.org/wiki/Mostafa_Ronaghihttp://en.wikipedia.org/wiki/P%C3%A5l_Nyr%C3%A9nhttp://en.wikipedia.org/wiki/P%C3%A5l_Nyr%C3%A9nhttp://en.wikipedia.org/wiki/P%C3%A5l_Nyr%C3%A9nhttp://en.wikipedia.org/wiki/Haemophilus_influenzaehttp://en.wikipedia.org/wiki/Haemophilus_influenzaehttp://en.wikipedia.org/wiki/Haemophilus_influenzaehttp://en.wikipedia.org/wiki/Hamilton_O._Smithhttp://en.wikipedia.org/wiki/Hamilton_O._Smithhttp://en.wikipedia.org/wiki/Hamilton_O._Smithhttp://en.wikipedia.org/wiki/Leroy_E._Hoodhttp://en.wikipedia.org/wiki/Leroy_E._Hoodhttp://en.wikipedia.org/wiki/Leroy_E._Hoodhttp://en.wikipedia.org/wiki/Leroy_E._Hoodhttp://en.wikipedia.org/wiki/Leroy_E._Hoodhttp://en.wikipedia.org/wiki/Leroy_E._Hoodhttp://en.wikipedia.org/wiki/Bacteriophage_%CF%86X174http://en.wikipedia.org/wiki/Bacteriophage_%CF%86X174http://en.wikipedia.org/wiki/Bacteriophage_%CF%86X174http://en.wikipedia.org/wiki/Bacteriophage_%CF%86X174


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DNA sequencing

Determination of nucleotide sequence

Two similar methods:

1. Maxam and Gilbert method

2. Sanger method They depend on the production of a mixture of

oligonucleotides labeled either radioactively orfluorescein, with one common end and differing inlength by a single nucleotide at the other end

This mixture of oligonucleotides is separated by highresolution electrophoresis on polyacrilamide gelsand the position of the bands determined


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The method developed by Maxam and Gilbert based onchemical modification of DNA and subsequent cleavage at

specific bases to generate a nested set of labeled fragments.

Also known as chemical sequencing, this method allowed

purified samples of double-stranded DNA to be used without

further cloning. While powerful and accurate, this method requires the use of

toxic chemicals.

This method's use of radioactive labeling and its technical

complexity discouraged extensive use after refinements in theSanger methods had been made.

This method originated in the study of DNA-protein

interactions (footprinting), nucleic acid structure and

epigenetic modifications to DNA, and within these it still has

important applications.

Maxam and Gilbert Method


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Maxam-Gilbert sequencing requires radioactive labeling at one 5'end of the DNA and purification of the DNA fragment to be

sequenced.

Chemical treatment then generates breaks at a small proportion

of one or two of the four nucleotide bases in each of four

reactions (G, A+G, C, C+T). The concentration of the modifying chemicals is controlled to

introduce on average one modification per DNA molecule.

Thus a series of labeled fragments is generated, from the

radiolabeled end to the first "cut" site in each molecule.

Recall that the fragments in the set increase in length one baseat a time from the 5 end of original labeled strand.

The fragments in the four reactions are electrophoresed side by

side in denaturing acrylamide gels for size separation.

Maxam and Gilbert Method


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Maxam and Gilbert MethodThe single stranded DNA fragment to be sequenced is end-labeled

by treatment with alkaline phosphatase to remove the 5phosphate It is then followed by reaction with P-labeled ATP in the presence

of polynucleotide kinase, which attaches P labeled to the5terminal

The labeled DNA fragment is then divided into four aliquots, eachof 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 residues

3. Aliquot C + Hydrazine, which modifies thymine + cytosine residues

4. Aliquot D + Hydrazine + 5 mol/l NaCl, which makes the reaction specific forcytosine

The four are incubated with piperidine which cleaves the sugar

phosphate backbone of DNA next to the residue that has beenmodified

To visualize the fragments, the gel is exposed to X-ray film forautoradiography, yielding a series of dark bands each correspondingto a radiolabeled DNA fragment, from which the sequence may beinferred.


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A methyl group is added to guanine, the modified base is

removed from its sugar by heating, and the exposed

sugar is removed from the backbone by heating in alkali.

To cleave at both A and G, the procedure is identical

except that a dilute acid is added after the methylation

step.

The reactions that cleave at C, or at C and T, involve

hydrazine to remove the bases and piperidine to cleave

the backbone.

The extent of the reaction can be carefully limited so

that, on average, only one G is evicted from each strand,

thus each strand is cleaved at only one of its guanine

sites.


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A radiolabeled strand to be sequenced and the fragmentscreated from that strand by a single cleavage at the site of G are

Each originalstrand is broken into a labeled fragment and an

unlabeled fragment.

All the labeled fragments start at the 5 end of the strand and

terminate at the base that precedes the site of a G along the

original strand.

Only the labeled fragments will be recorded once all the

fragments are separated on a gel and visualized by exposing thegel to an x-ray film to create an autoradiogram of the gel.


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Step 1: Preparation of Labeled Strands

Many copies of the DNA segment to be sequenced are

labeled with radioisotope 32P at the 5 end of the strand.

If the DNA is cloned in doublestranded form, then the 5

ends of both strands are labeled.

The DNA is then denatured, copies of one strand are

isolated from copies of the other strand, and each strand

is sequenced separately.


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Step 2: Generating a Nested Set ofLabeled Fragments

Copies of one labeled strand are divided into four

batches, and each batch is subjected to one of four

chemical cleavage reactions.

The reactions cleave the template strands at G, G and A,

C, or C and T, respectively.

All labeled fragments in each batch begin at the 5 end of

the original strand.


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Step 3: Electrophoresis and Gel Reading

The fragments from the four reactions are separated inparallel on four lanes of a gel by electrophoresis.

An autoradiogram of the gel shows the positions of the

labeled fragments only.

Each of the four lanes is labeled by the base or bases atwhich the original strand was cleaved.

Fragments cleaved at C show up in two lanes, the one

marked C and the one marked C and T.

Fragments cleaved at T are identified by noting that theyappear in the lane marked C and T, but do not appear in

the lane marked C.


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Fragments ending in A or G can be similarly identified.

Note that the fragment cleaved at the first base will not

show up on the gel, so the first base at the 5 end of the

original strand cannot be determined.

The band corresponding to the shortest fragments is at

the bottom of the autoradiogram.

The 5-to-3 sequence of the original strand is read by

noting the positions and lanes of the bands from the

bottom to the top of the autoradiogram.


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Frederick Sanger

Discovered DNA sequencing by chain

termination method

Nobel Prize 1 (1958)

Complete amino acid

sequence of insulin

Nobel Prize 2 (1980)

For DNA sequencing


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Sanger Method

Generates the nested set of labeled fragments from a

template strand by replicating the template strand to be

sequenced and interrupting the replication at one of the four

bases.

Four different replication reactions produce fragments that

terminate in A, C, G, or T, respectively. DNA synthesis using deoxy- and dideoxynucleotides that

results in termination of synthesis at specific nucleotides

Requires a primer, DNA polymerase, a template, a mixture of

nucleotides, and detection system Incorporation of dideoxynucleotides into growing strand

terminates synthesis

Synthesized strand sizes are determined for each

dideoxynucleotide rxn by using gel or capillary electrophoresis


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Dideoxynucleotide

no hydroxyl group at 3 end

prevents strand extension

CH2O

OPPP

5

3

BASE


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Dideoxy nucleotides

In the Sanger chain termination method, the nucleotide analog iscalled a dideoxynucleotide.

Are added in small proportion

When the correct amount is added to the solution, the chain will

be terminated at each occurrence of the complementary

nucleotide in the template because DNA polymerase cannot addanother base to the analog.

For example, if the right amount of dideoxy A is added, then the

chain will be terminated at each occurrence of T in the template.

To determine the complete sequence requires a separate

reaction for each of the four bases A, T, C, and G.

These strands are complementary to the template strand, and

terminate opposite the site of a T on the template strand.

Complementary strands terminating in either A, G, C, or T are

produced by the inclusion in the reaction mixture of ddATP,ddGTp, ddCTP, or ddTTP, respectively.


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The primer is essential to initiate replication of the templates

by DNA polymerase.

The most convenient method for adding a known sequence to

the 3 end of the template strand is to clone the strand in the

single stranded cloning vector Ml3 so that a known M13sequence will always flank the unknown DNA insert and can

serve as the site for binding a standard primer.

Also, the Ml3 cloning protocol automatically creates two types

of clones, each type containing a DNA insert whose sequence

is complementary to that of the other DNA insert.

Thus, the two complementary strands may be sequenced and

the two sequences cross-checked to ensure sequence

accuracy.


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DNA sequencing continued

In the dideoxy method of sequencing, the template DNAthat is to be sequenced is mixed with a primercomplementary to the template DNA and the four normaldNTPs, one of which is radioactively labeled forsubsequent visualization purposes.

This mixture is then splint into four different tubes thatare labeled A, C, G, and T. Each tube is then spikedwith a different ddNTP (ddATP for tube A, ddCTP fortube C, ddGTT for tube G, or ddTTP for tube T).

DNA polymerase is added and using the DNA templateand its complementary primer, the synthesis of newstrands of DNA complementary to the template begins.

Occasionally a dideoxynucleotide is added instead of thenormal deoxynucleotide and synthesis of that strand is

terminated at that point.


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In the tube containing ddATP, some percentage of newlysynthesized molecules will get a ddATP in each placethat there is a T in the template DNA.

The result is a set of new DNA molecules in tube A,

each of which ends in an A. A similar type of reaction occurs in the three other tubesto result in molecules that end in C, G, and T in tubes C,G, and T respectively.

After the synthesis reactions are complete, the products

of the four different tubes are loaded onto four adjacentlane of a polyacrylamide gel and the different fragmentsare separated by size.

The sequencing gel is able to resolve fragments thatdiffer in size from each other by only one base.


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After electrophoresis to separate the fragments by size,the fragments are visualized to exposing the gel tophotographic film (Remember that one nucleotide wasradioactively labeled).

All fragments in lane A will end in an A, fragments inlane C will all end in a C, fragments in lane G will all endin a G, and fragments in lane T will all end in a T.

The sequence of the DNA is read from the gel bystarting at the bottom and reading upward.


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Chain Termination


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Chain Terminator Basics

Target

Template-Primer

Extend

ddA

ddG

ddC

ddT

Labeled TerminatorsddA

AddC

ACddG

ACG ddT

TGCA

dN : ddN

100 : 1


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CCGTAC3 55 3primer

dNTP

ddATP

GGCA

ddTTP

GGCAT

ddCTP

GGC G

ddGTP

GGGGCATG

A T C G


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All Possible Terminations


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Sequence detection

To detect products ofsequencing reaction

Include labelednucleotides

Formerly, radioactivelabels used

Now, fluorescent labelsused

Use different fluorescenttag for each nucleotide

Can run all four bases insame lane

TAGCCACGTATCGAA*

TAGCCACGTATC*

TAGCCACG*

TAGCCACGT*


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Sequence separation

Terminated chains need tobe separated

Requires one-base-pairresolution

See difference betweenchain of X and X+1 base

pairs

Gel electrophoresis

Very thin gel High voltage

Works with radioactiveor fluorescent labels

A T C G

+


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Step 1: Template Preparation

Copies of the template strand are cloned in Ml 3.

They are thus flanked at their 3 ends by a known

sequence that will bind to a standard primer.


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Step 2: Generating a Nested Set ofLabeled Fragments

Copies of each template strand are divided into four batches, andeach batch is used for a different replication reaction.

Copies of the same standard primer and DNA polymerase is

used in all four reactions,

To synthesize fragments, all of which terminate at A, the dideoxy

analog ddATP is added to the reaction mixture along with dATP,dGTP, dCTP, dTTP the standard primer and DNA polymerase 1.

The ddATPs and one of the dNTPs are labeled with a radioactiveisotope to produce radio- Iabeled strands.

The figure shows a short template strand, the primer, the four

reaction mixtures, and the labeled strands produced by each

reaction.

Note that the synthesized fragments from the four reaction

mixtures compose the set of nested fragments needed to

determine the order of the bases in the strand complementary tothe tem late strand.


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Step 3: Electrophoresis and Gel Reading

The fragments from the four reaction mixtures are loaded

into four parallel lanes of a polyacrylamide gel and

separated by length using electrophoresis.

An autoradiogram of the gel is read as described in the

main text to determine the order of the bases in thestrand complementary to that of the template strand.

Again, since the bands corresponding to the shortest

fragments are at the bottom of the autoradiogram, the 5-

to-3 sequence of the strand complementary to thetemplate strand is read from the bottom to the top of the

autoradiogram.


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Polyacrylamide Gel Electrophoresis

Separates

fragments

based on size


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Dideoxy DNA Sequencing


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Sequencing of DNA by

the Sanger method


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DNA Sequencing 5.17)


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Shotgun Sequencing

Since only short stretches of DNA, several hundred to a

thousand base pairs in length, can be obtained from a

single sequencing gel, many shell sequences must be

generated separately and then combined to determine

the sequence of a much longer DNA fragment. Various strategies have been developed to generate

these short sequences from the larger fragment.

The shotgun approach is the most widely used in the

larger sequencing projects.


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Copies of a long fragment to be sequenced are broken

into much shorter fragments that overlap one another,

and the short fragments are cloned.

Those clones are then picked at random and sequenced.

The sequence of the long fragment is determined by

finding overlaps among the short sequences and

assembling those sequences into the most likely order.

Numerous computer algorithms have been developed to

facilitate the assembly of long sequences.

Shotgun Sequencing


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Inevitably, gaps remain in the sequence of the long fragment, and

they are filled by switching to a directed sequencing strategy.

That is, the short clones are no longer sequenced at random, but

rather, short sequences at the end of a continuous stretch of known

sequence provide the information necessary to construct a probe to

pick out a clone, or region of a clone, whose sequence will extendthe known sequence.

Most of the large sequencing projects to date have used a mixture of

random and directed sequencing strategies to complete the

sequence of long, contiguous stretches of DNA.

The advantage of the random, or shotgun, strategy is that in the

course of picking clones at random and sequencing them, any given

region is usually sequenced many times, thereby reducing the errors

in the final sequence.

Shotgun Sequencing


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Automated DNA sequencing

In automated DNA sequencing a radioactivedeoxynucleotide is not used and all four dideoxy reactionsare done in a single tube.

This is possible because each ddNTPs is labeled with adifferent flourescent dye.

Therefore the dye present in each synthesized fragmentcorresponds to the dye attached to the dideoxynucleotidethat was added to terminate the synthesis of that particularfragment.

The contents of the single tube reaction are loaded onto a

single lane of a gel and electrophoresis is done. A flourimeter and computer are hooked up to the gel and

they detect and record the dye attached to the fragments

as they come off the gel.

The sequence is determined by the order of the dyes

coming off the gel.


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Automated DNA sequencing

Documents

Methods of DNA Sequencing