Dna Sequencing(Sanger's Method)

7/29/2019 Dna Sequencing(Sanger's Method)

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Determining DNA Sequence

Originally 2 methods were invented around 1976, butonly one is widely used: the chain-termination methodinvented by Fred Sanger. The other method is Maxam-Gilbert chemical degradation method,

which is still used for specialized purposes, such as analyzingDNA-protein interactions.

More recently, several cheaper and faster alternatives have been invented. It is

hard to know which of these methods, or possibly another method, will ultimatelybecome standard. We will discus two of them: 454 pyrosequencing andIllumina/Solexa sequencing


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Sanger Sequencing Uses DNA polymerase to synthesize a second

DNA strand that is labeled. DNA polymerasealways adds new bases to the 3 end of a primerthat is base-paired to the template DNA.

DNA polymerase is modified to eliminate itsediting function

Also uses chain terminator nucleotides: Uses

special enzymes to synthesize fragments of DNA thatterminate when a selected base appears in the stretchof DNA being sequenced,Dideoxy Nucleotides(ddNTPs), which lack the -OH group on the 3'carbon of the deoxyribose. When DNApolymerase inserts one of these ddNTPs into thegrowing DNA chain, the chain terminates, as

nothing can be added to its 3' end.


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Sequencing Reaction The template DNA is usually single stranded

DNA, which can be produced from plasmid

cloning vectors that contain the origin ofreplication from a single strandedbacteriophage such as M13 or fd. The primer iscomplementary to the region in the vectoradjacent to the multiple cloning site.

Sequencing is done by having 4 separatereactions, one for each DNA base.

All 4 reactions contain the 4 normal dNTPs, but

each reaction also contains one of the ddNTPs. In each reaction, DNA polymerase starts

creating the second strand beginning at theprimer.

When DNA polymerase reaches a base forwhich some ddNTP is present, the chain willeither: terminate if a ddNTP is added, or:

continue if the corresponding dNTP isadded. which one happens is random, based on

ratio of dNTP to ddNTP in the tube. However, all the second strands in, say, the A

tube will end at some A base: you get acollection of DNAs that end at each of the A's inthe region being sequenced.


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Electrophoresis The newly synthesized DNA from

the 4 reactions is then run (inseparate lanes) on anelectrophoresis gel.

The DNA bands fall into a ladder-like sequence, spaced one baseapart. The actual sequence canbe read from the bottom of the gel

up. Automated sequencers use 4 differentfluorescent dyes as tags attached to thedideoxy nucleotides and run all 4 reactionsin the same lane of the gel. Todays sequencers use capillary

electrophoresis instead of slab gels. Radioactive nucleotides (32P) are used

for non-automated sequencing.

Sequencing reactions usuallyproduce about 500-1000 bp ofgood sequence.


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DNA sequencing example

Problem Statement:Consider the following DNAsequence (from firefly luciferase). Draw the sequencing

gel pattern that forms as a result of sequencing the

following template DNA with ddNTP as the capper.

atgaccatgattacg...

Solution:

Given DNA template: 5'-atgaccatgattacg...-3'

DNA synthesized: 3'-tactggtactaatgc(ddCTP)...-5'


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DNA sequencing exampleGiven DNA template: 5'-atgaccatgattacg...-3'

DNA synthesized: 3'-tactggtactaatgc...-5'

Gel pattern: +-------------------------+

lane ddATP - | W | | || |

lane ddTTP - | W | | | | | |

lane ddCTP - | W | | | |

lane ddGTP - | W || | |

+-------------------------+

Electric Field +Decreasing size

where "W" indicates the well position, and "|"

denotes the DNA bands on the sequencing gel.


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This picture is a radiograph. The dark color of the lines is

proportional to the radioactivity from 32P labeled adenonsine

in the transcribed DNA sample.


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Reading a sequencing gel+ Beginning from the right i.e. , the smallest DNA fragments.

+The sequence read will be read in the 5'-3' direction.This sequence will be exactly the same as the RNA that

would be generated to encode a protein.

+As an example, in the problem given, the smallest DNA fragment

on the sequencing gel is in the C lane, so the first base is a C. Thenext largest band is in the G lane and so on.

Therefore the sequence of the first two bases is CG.

The sequence of the first 30 or so bases of the DNA are:

CGTAATCATGGTCATATGAAGCTGGGCCGGGCCGTGC....When this is made as RNA, its sequence would be:

CGUAAUCATGGUCAUAUGAAGCUGGGCCGGGCCGUGC....Note that the information content is the same, only the T's have

been replaced by U's!.


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Translating the DNA sequence

The order of amino acids in any protein is specified by the

order of nucleotide bases in the DNA.

Each amino acid is coded by the particular sequence of three bases.

To convert a DNA sequence

First, find the starting codon. The starting codon is always

the codon for the amino acid methionine. This codon isAUG in the RNA (or ATG in the DNA):

GCGCGGGUCCGGGCAUGAAGCUGGGCCGGGCCGUGC....

Met

In the given example the next codon is AAG. The first base(5'end) is A, so that selects the 3rd major row of the table. The

second base (middle base) is A, so that selects the 3rd column of

the table. The last base of the codon is G, selecting the last line in

the block of four.


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The codon table

5-Base Middle Base 3-BaseU(=T) C A G

U(=T) Phe Ser Tyr Cys U(=T)

Phe Ser Tyr Cys C

Leu Ser Term Term A

Leu Ser Term Trp G

C Leu Pro His Arg U(=T)Leu Pro His Arg C

Leu Pro Gln Arg A

Leu Pro Gln Arg G

A Ile Thr Asn Ser U(=T)

Ile Thr Asn Ser C

Ile Thr Lys Arg A

Met Thr Lys Arg G

G Val Ala Asp Gly U(=T)

Val Ala Asp Gly C

Val Ala Glu Gly A

Val Ala Glu Gly G


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Translating the DNA sequence

This entry AAG in the table is Lysine (Lys).

Therefore the second amino acid is Lysine.

The first few residues, and their DNA sequence, are as follows

(color coded to indicate the correct location in the

codon table):Met Lys Leu Gly Arg ...

AUG AAG CUG GGC CGG GCC GUG C..

This procedure is exactly what cells do when they synthesizeproteins based on the mRNA sequence. The process of translation

in cells occurs in a large complex called the ribosome.


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Automated procedure for DNAsequencing

A computer read-out of the gel generates a false color image

where each color corresponds to a base. Then the intensities are

translated into peaks that represent the sequence.

Dna Sequencing(Sanger's Method)

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