DNA SEQUENCING

Chemical Modification Method

Chain Termination Method
 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
 Sequencing Methods
• To determine the order of the nucleotide
bases adenine, guanine, cytosine, and
thymine in a molecule of DNA two methods
were used
1. Maxam and Gilbert; Chemical Sequencing
2. Sanger; Chain Termination Sequencing
• These two are conventional methods
• Robotics and automated sequencing are
based on these methods
Advanced methods:

1.Short Gun Sequencing

2.Bridge PCR Sequencing
Next generation methods:

1. Massively parallel signature

sequencing
2. Polony sequence
3. Pyrosequencing
4. Illumina sequencing
5. Solid sequencing
6. DNA nanoball sequencing.
 The Need for DNA Sequencing
• Gene isolation
• Sequence charaterization
• Forensics
• Molecular Archeology
• Gene Gene Interaction
• Gene Protein Interaction
• Cloning
 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 sequenced
III. Chemical treatment generates breaks in DNA
IV. 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′

32
P 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
 Maxam Gilbert Sequencing

DMS FA H H+S

G G C C
G A T C
G G T
G G C C
C
A T
G C
A C
A T

P 5′ G A C G T G C A A C G A 3′
32
 Maxam And Gilbert Sequencing:
10 nucleotide DNA sequence:
5’P-TTCAGCCGAT-OH3’
First step:

5’P-TTCAGCCGAT-OH3’+H2O 5’OH-TTCAGCCGAT-OH3’+Pi

5’OH-TTCAGCCGAT-OH3’+A-P-P-32P 5’32P-TTCAGCCGAT OH3’+ADP

gamma-32P]ATP

The DNA solution is divided into four aliquots:

1. G only
2. G+A
3. C+T
4. C only

G only G+A C+T C

only
1. G only :
- In this tube the DNA is incubated with dimethyl sulfate (DMS).
- 5’32P-TTCA and 5’32P-TTCAGCC- two G residue present.
- one strand will be 4 nucleotide other will be 7 nucleotide long.

2. A+G :
- Here the DNA is protonated rather than methylated.
- TTC, TTCA, TTCAGCC, TTCAGCCG.
- measuring 3,4 ,7 and 8 nucleotides in length.

3. C+T :
- DNA is reacted with hydrazine(NH2-NH2) and this followed with
piperidine treatment.
- T, TT, TTCAG, TTCAGC, TTCAGCCGA.
- measuring 1, 2, 5, 6 and 9 nucleotide in length
4. C only :

- If hydrazine treatment is carried out in presence of

1.5M NaCl.
- TT, TTCAG, TTCAGC.
- measuring 2, 5,and 6 nucleotides long.
Next
steps:
- The four differently fragmented sample of DNA are
simultaneously electrophoresed in parallel lanes on a
sequencing gel

- After electrophoresis gel is exposed to a photographic film

- The sequence of DNA simply read of f this autoradiogram

SUMMARY
………
Maxam Gilbert Sequencing: Process Summarized

1. Label 5’- end of DNA

2. Aliqot DNA sample in 4 tubes
3. Perform base modification reaction
4. Perform Cleavage reaction
5. Perform Gel Electrophoresis
6. Perform Autoradiography
7. Interpret results
 Sanger; Chain Termination Sequencing
• It is PCR based method
• A modified DNA replication reaction
• Developed in MID-1970 by TWO scientists
SANGER and A.R.COULSON
• It is an ENZYMATIC method
• PRINCIPLE:
• Use of DIDEOXY NUCLEOSIDE
TRIPHOSPHATES (ddNTP) as DNA Chain
terminators
 Difference between d NTP & dd NTPs:

• A ddNTP is a laboratory made chemical molecule

which is act as a ANALOGUE to dNTP

•Its LACKS the HYDROXYL group at both the 2’

and 3’ carbons of the sugar

•Significance of 3’ hydroxyl group– Formation of

phosphodiester bond
The 3′-OH group necessary for formation of the phosphodiester bond is missing in ddNTPs
 REQUIRMENTS:
• ssDNA Fragment
• DNA Primer
• DNA Polymerase
• All FOUR DEOXY RIBONUCLEOSIDE TRIPHOSPHATES
• Small con. Of the DI-DEOXY NUCLEOSIDES TRI-PHOSPHATES
or ddNTP
Ex: dd ATP
dd GTP
dd CTP & dd TTP
• DNA sequencing is carried out in four reaction tubes in 4 steps

– STEP 1: Denaturation

– Step 2: Primer attachment and extension of bases

– Step 3: Termination

– Step 4: Poly acrylamide gel electrophoresis

Sanger; Chain Termination Sequencing
A G C T G C C C G
ddATP + ddA
A
four dNTPs dAdGdCdTdGdCdCdCdG

ddCTP + dAdGddC
C
four dNTPs dAdGdCdTdGddC
dAdGdCdTdGdCddC
dAdGdCdTdGdCdCddC

ddGTP + dAddG
G four dNTPs dAdGdCdTddG
dAdGdCdTdGdCdCdCddG

T
ddTTP + dAdGdCddT
four dNTPs dAdGdCdTdGdCdCdCdG
 Chain Termination Sequencing

3′
G
G A T C G
Longer fragments T
A
ddG
A
A
T
C
Shorter fragments A
ddG T
G
5′

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

 Sanger Sequencing: An Example
5’-TACACGATCGA-3’
3’-ATGTGCTAGCT-5’

Denature the sequence

Use only forward primer i.e. using 3’-5
 Amplification in ddTTP Amplification in ddATP

3’-ATGTGCTAGCT-5’
3’-ATGTGCTAGCT-5’
5’-TA-3’
5’-T-3’ 5’-TACA-3’
5’-TACACGAT-3’ 5’-TACACGA-3’
5’-TACACGATCGA-3’
Amplification in dGTTP Amplification in ddCTP

3’-ATGTGCTAGCT-5’ 3’-ATGTGCTAGCT-5’
5’-TACACG-3’ 5’-TAC-3’
5’-TACACGATCG-3’ 5’-TACAC-3’
5’-TACACGATC-3’
 Reading Sequence
BAND ddTTP ddATP ddGTP ddCTP 3’ 3’
12 bp
11 bp
10 bp
9 bp
8 bp
7 bp
6 bp
5 bp
4 bp
3 bp
2 bp
1 bp
5’ 5’
 Sanger Sequencing: Process Summarized

1. Get enough quantity of DNA (Run PCR)

2. Aliqot DNA into four different tubes
3. Prepare PCR reaction mix as below:
• Primer, taq PM, template(ss DNA), dNTPS (All)
and ddNTPs(ddATP, ddGTP,ddCTP & ddTTP
respectively)
4. Run PCR
5. Perform Gel Electrophoresis
6. Interpret results
 Sanger Method Automated fluorescent DNA
sequencing method
Primer is radio labelled with ddNTP labelled with 4
either 32p or 35s different FLUORESCENT
dyes
There is only a single round of Hybridization, Synthesis and
DNA synthesis Denaturation is repeated many
times
Sequences are carried out in 4 Four chain-terminated
reaction tubes products are run on the same
tube
300bp 500bp to 100,000bp
 Pyro sequencing

• DNA Sequencing based on the “SEQUENCING BY SYNTHESIS”

• Its relies on the detection of PYROPHOSPHATE release on

NUCLEOTIDE incorporation, rather than CHAIN TERMINATION
• The single-strand DNA template is hybridized to a
sequencing primer and incubated with the enzymes

• The pyrosequencing method is based on detecting the

activity of DNA polymerase with another
chemiluminescent enzyme

• DNA polymerase
• ATP sulfurylase
• Luciferase and apyrase
• Substrates adenosine 5´ phosphosulfate (APS) and luciferin
 Applications of dna sequencing:
• To Find Genes
• Information regarding MUTATIONS
• Gene overlapping
• Identification of POLYMORPHSIMS
• Profiling of the DNA methylation in the genomes
• Exome sequencing
• Identification of GENE REGULATORY CONTROL
SEQUENCES
• To determine the PATERNITY of a child
 REFERENCES:
• text book of biochemistry by d.m.vasudevan
• Bio physical chemistry by upadhyaya and nath
• tietz textbook of clinical chemistry
• molecular biology— burtan .e. tropp
• Wilson and Walker Principles and techniques of Biochemistry
• Cell and molecular biology– e.d.p.de Roberti's 8 th edition
• Bioinformatics– practical approach– shui Qing ye
• Textbook of Biochemistry by u SatyaNarayana
• http://en.wikipedia.org/wiki/DNA_sequencing --Pyro sequencing
• DNA sequencing Written by: Anthony J.F. Griffiths