Its a presentation about DNA sequencing by basic methods and one of the advanced method along with applications of the dna sequencing.

2. DNA SEQUENCING
It is the process of determining the precise order of nucleotide within
a DNA molecule.
First DNA sequencing is obtained in the early 1970,by academic researches using
laborious method based two dimensional chromatography.
Four canonical bases :
• Adenine
• Guanine
• Thymine
• Cytosine

3. History
• 1953 - structure of DNA established as a double helix.
• RNA sequencing was one of the earliest forms of
nucleotide sequencing.
• 1970 - first method of DNA sequencing involved a location
specific primer extension strategy.
• 1977 - Frederick sanger published a method for DNA
sequencing with chain terminating inhibitors.

4. • 1977 - Allan Maxam and Walter Gilbert developed
DNA sequencing by chemical degradation.
• 1977 - the first genome to be sequenced was that of
bacteriophage φX174.
• 1990 - several new methods are developed in the mid
to late 90’s.
• 2003 - Complete Human Genome Project.

5. Methods of DNA sequencing Basic
methods:
1. Maxam-Gilbert sequencing
2. Chain termination method
Advanced methods:
1.Short Gun Sequencing
2.Bridge PCR Sequencing

6. Next generation methods:
1. Massively parallel signature sequencing
2. Polony sequence
3. Pyrosequencing
4. Illumina sequencing
5. Solid sequencing
6. DNA nanoball sequencing.

7. 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

8. 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

9. 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
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.

10. SUMMARY………

11. THE SANGER CHAIN - TERMINATION METHOD
•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

12. 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

13. 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

14. •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

16. Sanger Method Automated fluorescent DNA
sequencing method
Primer is radio labelled with either
32p or 35s
ddNTP labelled with 4 different
FLUORESCENT dyes
There is only a single round of DNA
synthesis
Hybridization, Synthesis and
Denaturation is repeated many
times
Sequences are carried out in 4
reaction tubes
Four chain-terminated products are
run on the same tube
300bp 500bp to 100,000bp

17. 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

18. •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

20. 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

21. 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 8TH 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