Sequencing

What is Sequencing A lab technique used to find out the

sequence of nucleotide bases in a DNA

molecule or fragment.

It is a deciphering of the exact order of

base sequence in a nucleotide

Sequence.

Examples are dideoxy sequencing and

maxam-gilbert sequencing.

Method of the Sequencing There are two methods of sequencing

Maxam and Gilbert method (the manual or

chemical sequencing )

And

Sanger method using dideoxynucleotide

(modern sequencing)

Sanger method is more efficient and uses fewer toxic chemicals and lower amounts of radioactivity than the method of Maxam and Gilbert, it rapidly became the method of choice.

DNA SEQUENCING

Principles & methods

Topic Outline

Principles of DNA Sequencing

Methods of DNA Sequencing

Principles of DNA Sequencing

Cycle sequencing (one cycle)

Denaturation

Annealing Extension/ termination

Partial copies of DNA fragments made with DNA polymerase

Collection of DNA fragments that terminate with A,C,G or T using ddNTP or chemical.

Separate by gel electrophoresis

Read DNA sequence

Components For Reaction DNA fragment to be sequenced

DNA Template

Oligonucleotide Primer

Enzyme: DNA Polymerase

Deoxynucleotide triphosphate: dNTPs [dATP,

dTTP, dCTP, dGTP]

Dideoxy nucleotides triphosphate (ddNTP):

[ddA, ddC, ddG and ddT]

Radioactive label

Denaturation, Gel electrophoresis

Four aliquots – treated with chemicals ( modification)

Cleaved by piperidine

Separated by Electrophoresis

Add in DNA polymerase I,

dNTPs, ddNTPs

Extension /synthesis of complementary

strands

Synthesis termination

Dead-end product

Run gel divide by size

DNA sequencing by the Sanger method

The standard DNA sequencing technique is the Sanger method,

named for its developer, Frederick Sanger, who shared the 1980

Nobel Prize in Chemistry. This method begins with the use of

special enzymes to synthesize fragments of DNA that terminate

when a selected base appears in the stretch of DNA being

sequenced. These fragments are then sorted according to size

by placing them in a slab of polymeric gel and applying an

electric field -- a technique called electrophoresis. Because of

DNA's negative charge, the fragments move across the gel toward

the positive electrode. The shorter the fragment, the faster it

moves. Typically, each of the terminating bases within the

collection of fragments is tagged with a radioactive probe for

identification.

DNA sequencing example

Problem Statement: Consider the following DNA

sequence (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...-5'

DNA sequencing example

Given 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.

Running the reaction of all the dideoxy nucleotides

using different dyes generates this type of diagram in same lane.

• Sometimes the spacing between the bands is hard to measure.

• Thus use machine to run and read the electrophoresis.

• Capillary electrophoresis: the fragments are piped through a tiny glass-fiber capillary during the electrophoresis step, and they come out the far

end in size-order.

Chemical cleave method

Sequence small fragments of DNA

The radioactive labelling is done on the dsDNA.

Division of aliquots is done by methylation or removal of base.

Requires DNA

Breaks DNA at different nucleotides

Enzymatic cleave method

Sequencing small fragments are problematic.

The radioactive labelling is done on the ssDNA.

Allow high throughput automated sequencing techniques.

Allow Real Time detection.

Requires DNA synthesis

Termination of chain elongation

• Enzymatic plus-minus method by Sanger - 1975 - using DNA polymerase

• chemical degradation method by Maxam and Gilbert - 1977

• Sanger DNA sequencing method using polyacrylamid gels

• semiautomated methods for running and analysing sequencing gels

• automated methods like: PCR cycle sequencing or fluorescent DNA sequencing

• DNA phage X174, 5386 bp (plus-minus method)

• SV40 DNA 5243 bp (Maxam-Gilbert method)

• recombinant plasmid pBR322, 4362 bp (Maxam-Gilbert method)

• cloning experiments

• PCR products can be sequenced directly

• chromosome mapping

• genome sequencing projects

• generating protein information

Comparison of the DNA Sequencing Methods

Maxam-Gilbert Method Sanger Method

Manual sequencing Automatic sequencing

(Chain-termination

method)

More effort needed in

running gels

Uses automated technology

Time consuming Easier, faster (1 lane per

sample instead of 4)

Radioactivity is used to label

the fragments

Uses special fluorescent dyes

to label the DNA

fragments

1

2

3

4

A sequencing gel

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.

The codon table

5’-Base Middle Base 3’-Base

U(=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

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 synthesize

proteins based on the mRNA sequence. The process of translation

in cells occurs in a large complex called the ribosome.

Automated procedure for DNA sequencing

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.

High-throughput seqeuncing: Capillary electrophoresis

The human genome project

has spurred an effort to

develop faster, higher

throughput, and less

expensive technologies

for DNA sequencing.

Capillary electrophoresis

(CE) separation has many

advantages over slab gel

separations. CE separations are faster and are capable of producing

greater resolution. CE instruments can use tens and even

hundreds of capillaries simultaneously. The figure show a simple

CE setup where the fluorescently-labeled DNA is detected as it

exits the capillary.

Laser

PMT

Focusing

lens

Sheath flow cuvette

Sheath flow

Collection Lensc Collection Lensc

Beam block

filter

DNA sequencing. • Dideoxy analogs of normal nucleotide triphosphates (ddNTP)

cause premature termination of a growing chain of nucleotides.

ACAGTCGATTG

ACAddG

ACAGTCddG

ACAGTCGATTddG

• Fragments are separated according to their sizes in gel electrophoresis. The lengths show the positions of “G” in the original DNA sequence.

Nucleotides and phosphodiester bond.

Phosphodiester bond

Sequencing cDNA libraries.

• mRNA is pooled from the tissues which express genes.

• cDNA libraries are prepared by copying of mRNA with reverse transcriptase.

• Expressed Sequence Tags (EST) – partial sequences of expressed genes.

• Comparing translated ESTs to annotated proteins – annotation of genes.