DNA SEQUENCINGChemical Modification Method

Chain Termination MethodVeerendra Singh Nagoria

Assistant Professor Biotechnology

Objectives

• What is DNA Sequencing ?

• History of development

• Basic Methods- Chain

termination and Chemical

modification method

What is DNA Sequencing ?

• Determining the precise order of nucleotides in DNA.

• We need to determine the order of nucleotide bases in a strand of DNA for sequencing.

The Need for DNA Sequencing

• Gene isolation• Sequence charaterization• Forensics• Molecular Archeology• Gene Gene Interaction• Gene Protein Interaction• Cloning

DNA • Deoxyribonucleic Acid• Stores genetic information• Four different nucleotides A,T,G,C• DNA comprises of a long molecule analogous to a chain,

while the links of the chain are called Nucleotides

Historical Timeline

1870 – Miescher discovers DNA

1940 - Avery: Proposes DNA as ‘Genetic Material’

1953 – Watson & Crick “double helical structure”

1970 - Wu: Sequences λ Cohesive End DNA

1977 – Sanger: Dideoxy Chain Termination

1977 – Gilbert: Chemical Degradation

1986 – Partial Automation

1990 – Cycle Sequencing, Improved Sequencing Enzymes,

Improved fluorescent detection schemes

2002 – NGS: 454 , pyro sequencing

Cost per Genome

Cost per Megabases

Cost Data (source NHGRI)

Date Cost per Mb Cost per Genome Sep-01 $5,292.39 $95,263,072Mar-02 $3,898.64 $70,175,437Sep-02 $3,413.80 $61,448,422Mar-03 $2,986.20 $53,751,684Oct-03 $2,230.98 $40,157,554Jan-04 $1,598.91 $28,780,376Apr-04 $1,135.70 $20,442,576Jul-04 $1,107.46 $19,934,346Oct-04 $1,028.85 $18,519,312Jan-05 $974.16 $17,534,970Apr-05 $897.76 $16,159,699Jul-05 $898.90 $16,180,224Oct-05 $766.73 $13,801,124Jan-06 $699.20 $12,585,659Apr-06 $651.81 $11,732,535Jul-06 $636.41 $11,455,315Oct-06 $581.92 $10,474,556Jan-07 $522.71 $9,408,739Apr-07 $502.61 $9,047,003Jul-07 $495.96 $8,927,342Oct-07 $397.09 $7,147,571Jan-08 $102.13 $3,063,820Apr-08 $15.03 $1,352,982

Date Cost per Mb Cost per Genome Oct-08 $3.81 $342,502Jan-09 $2.59 $232,735Apr-09 $1.72 $154,714Jul-09 $1.20 $108,065Oct-09 $0.78 $70,333Jan-10 $0.52 $46,774Apr-10 $0.35 $31,512Jul-10 $0.35 $31,125Oct-10 $0.32 $29,092Jan-11 $0.23 $20,963Apr-11 $0.19 $16,712Jul-11 $0.12 $10,497Oct-11 $0.09 $7,743Jan-12 $0.09 $7,666Apr-12 $0.07 $5,901Jul-12 $0.07 $5,985Oct-12 $0.07 $6,618Jan-13 $0.06 $5,671Apr-13 $0.06 $5,826Jul-13 $0.06 $5,550Oct-13 $0.06 $5,096Jan-14 $0.04 $4,008Apr-14 $0.05 $4,920

Sequencing Methods• To determine the order of the nucleotide

bases adenine, guanine, cytosine, and thymine in a molecule of DNA two methods were used1. Maxam and Gilbert; Chemical Sequencing2. Sanger; Chain Termination Sequencing

• These two are conventional methods• Robotics and automated sequencing are

based on these methods

Maxam and Gilbert Method• In 1976–1977, Allan Maxam and Walter Gilbert

developed a DNA sequencing method based on chemical modification of DNA and subsequent cleavage at specific bases

I. Chemical Modification of DNA; radioactive labeling at one 5' end of the DNA (typically by a kinase reaction using gamma-32P ATP)

II. Purification of the DNA fragment to be sequencedIII. Chemical treatment generates breaks in DNAIV. Run on the gel

Chemical Modification and Cleavage

• Ploy nucleotide Kinase radioactive label at one 5' end of the DNA using gamma-32P

5 ′ G A C G T G C A A C G A A 3′

32P 5 ′ G A C G T G C A A C G A A 3′

Chemical Modification and Cleavage

• Base Modification using Dimethyl sulphate– Purine• Adenine• Guanine

– Only DMS------- G– DMS+ Formic acid-------G+A

• Cleavage of Sugar Phosphate backbone using Piperidine

Chemical Modification and Cleavage

• Base modification using Hydrazine– Pyrimidine• Cytocine• Thymidine

– Hydrazine----- C+T– Hydrazine + NaCl--------C

• Cleavage of Sugar Phosphate backbone using Piperidine

DMS

G

GG

G

FA

GA

GG

AG

AA

H

CTT

C

TC

CT

H+S

CC

CC

Maxam Gilbert Sequencing

32P 5 ′ G A C G T G C A A C G A 3′

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

G G+A T+C C

3′AGCAACGTGCAG5′

Longer fragments

Shortest fragments

G

A

Maxam-Gilbert Sequencing

32P 5 ′ G A C G T G C A A C G A 3′

Maxam Gilbert Sequencing: Process Summarized

1. Label 5’- end of DNA 2. Aliqot DNA sample in 4 tubes3. Perform base modification reaction4. Perform Cleavage reaction5. Perform Gel Electrophoresis6. Perform Autoradiography7. Interpret results

Sanger; Chain Termination Sequencing

• It is PCR based method• A modified DNA replication reaction• Growing chains are terminated by

dideoxynucleotides

The 3 -OH group necessary for formation of the phosphodiester bond is missing in ddNTPs′

ddATP + ddAfour dNTPs dAdGdCdTdGdCdCdCdG

ddCTP + dAdGddCfour dNTPs dAdGdCdTdGddC

dAdGdCdTdGdCddC dAdGdCdTdGdCdCddC

ddGTP + dAddGfour dNTPs dAdGdCdTddG

dAdGdCdTdGdCdCdCddG

ddTTP + dAdGdCddTfour dNTPs dAdGdCdTdGdCdCdCdG

A

C

G

T

Sanger; Chain Termination Sequencing

A G C T G C C C G

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

G A T C

3′GGTAAATCATG5′

Longer fragments

Shorter fragmentsddG

ddG

Chain Termination Sequencing

Sanger Sequencing: An Example

5’-TACACGATCGA-3’3’-ATGTGCTAGCT-5’

Denature the sequenceUse only forward primer i.e. using 3’-5

3’-ATGTGCTAGCT-5’5’-T-3’5’-TACACGAT-3’

Amplification in ddTTP

3’-ATGTGCTAGCT-5’5’-TA-3’5’-TACA-3’5’-TACACGA-3’5’-TACACGATCGA-3’

Amplification in ddATP

Amplification in dGTTP Amplification in ddCTP

3’-ATGTGCTAGCT-5’5’-TACACG-3’5’-TACACGATCG-3’

3’-ATGTGCTAGCT-5’5’-TAC-3’5’-TACAC-3’5’-TACACGATC-3’

Reading SequenceBAND ddATP ddTTP ddGTP ddCTP12 bp

11 bp

10 bp

9 bp

8 bp

7 bp

6 bp

5 bp

4 bp

3 bp

2 bp

1 bp

3’

5’ 5’

3’

Sanger Sequencing: Process Summarized

1. Get enough quantity of DNA (Run PCR)2. Aliqot DNA into four different tubes3. Prepare PCR reaction mix as below:• Primer, taq PM, template(ss DNA), dNTPS (All)

and ddNTPs(ddATP, ddGTP,ddCTP & ddTTP respectively)

4. Run PCR5. Perform Gel Electrophoresis6. Interpret results

Thank You

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