A short, crisp PPT on the methods of DNA Seq.

1. DNA
Sequencing
DNA Sequencing

2. S.No Contents
1. DNA Sequencing
2. History of DNA Sequencing
3.
Steps in DNA Sequencing
A. Sample Preparation
B. PCR Amplification of Target Sequencing
C. Amplicons Purification
D. Sequencing Pre-preparation
E. DNA Sequencing
F. Data Analysis
4. The Different Methods
5. How Does Sangers Sequencing work?
6. Steps of Sangers Sequencing
7. Challenges
8. Limitations of DNA Sequencing
9. Applications of DNA Sequencing
10. Conclusion

3. DNA Sequencing :
 The process of determining the sequence of determining the nucleic acid sequence – the order of
nucleotides in DNA
 Determines the order of the four bases: Adenine, Guanine, Cytosine and Thymine
 Accelerated biological and medical research and discovery
 Uses: medical diagnosis, biotechnology, forensic biology, virology

4. History:
 The first full DNA genome - bacteriophage φX174 (Virus) (1977) – Chemical Degradation method
 By 1990 - the U.S. trials on
 Mycoplasma capricolum,
 Escherichia coli, (40 Lakhs)
 Caenorhabditis elegans, at a cost of US$0.75 per base (=55Rs)
 Saccharomyces cerevisiae
 In 1995 - the first complete genome of a free-living organism, Haemophilus influenzae.
(1,830,137 bases)
 1977 – Fredrick Sanger postulated the first time method for sequencing the DNA, named as Chain
Termination Method.

6. Different Methods:
1. Maxam and Gilbert Method
2. Chain Termination Method (Sangars Method)
3. Semi- automated method
4. Automated Method
5. Pyro-sequencing
6. Whole Genome shot gun Sequencing method
7. Clone by the clone sequencing method
8. Next-generation sequencing method

7. Sangars Sequencing
 1977 – Fredrick Sangar and colleagues
 Based on - selective incorporation of chain terminating
dideoxynucleotides by DNA polymerase during in-vitro DNA replication.

8. 1
2

9.  A DNA primer complementary to the template DNA is the starting point for DNA synthesis.
 In the presence of the four deoxynucleotide triphosphates (dNTPs: A, G, C, and T), the
polymerase extends the primer by adding the complementary dNTP to the template DNA
strand.
 To determine which nucleotide is incorporated into the chain of nucleotides, four
dideoxynucleotide triphosphates (ddNTPs: ddATP, ddGTP, ddCTP, and ddTTP) labeled with
a distinct fluorescent dye are used to terminate the synthesis reaction.
 Compared to dNTPs, ddNTPs has an oxygen atom removed from the deoxyribonucleotide,
hence cannot form a link with the next nucleotide. (Principle)
Short Introduction

10. Steps of Sangars Sequencing Method
(1) The double-stranded DNA (dsDNA) is denatured into two single-stranded DNA (ssDNA).
(2) A primer that corresponds to one end of the sequence is attached.
(3) Four polymerase solutions with four types of dNTPs but only one type of ddNTP are
added.
(4) The DNA synthesis reaction initiates and the chain extends until a termination
nucleotide is randomly incorporated.
(5) The resulting DNA fragments are denatured into ssDNA.
(6) The denatured fragments are separated by gel electrophoresis and the sequence is
determined.

15. 1 2 3 4 1

17. Polyacrylamide Gel Electrophoresis Plate
A T G C

18. Sequence Reading
AGCTGATAGGCACTTATGG
Hence, the DNA is sequenced…

20. Challenges
 Poor quality in the first 15-40 bases of the sequence due to primer binding.
 Deteriorating quality of sequencing traces after 700-900 bases.
 In cases where DNA fragments are cloned before sequencing, the resulting sequence may contain parts of
the cloning vector.
 Current methods can directly sequence only relatively short (300-1000 nucleotides long) DNA fragments in a
single reaction.

21. Limitations
 A sequencing platform is needed. For that, a huge, high-speed supercomputer
is required.
 Also, several sequences like, repetitive DNA, fragmented genes, and other
duplicated regions can not be studied properly.
 The chance of errors in the pre-sample processing can cause big economical
loss.

23. Applications of DNA Sequencing
1. Forensic identification - clearer than fingerprints so possible to find criminals
2. (DNA evidence (blood, skin or hair) can be matched to the DNA of a suspect to determine information about
where an individual was and who they may have come in contact with. ( Important in cases of rape. )
3. Screen for certain genetic diseases or risk factors.
4. Information about an embryo before it is implanted.
5. Disaster victim identification - plane crash, fire or terrorist attack.
6. DNA testing is also more reliable than eyewitness.

24. Applications
 Study of genomes and the proteins they encode to identify potential drug targets.
 To study how different organisms are related and how they evolved.
 Identification of organisms present in a body of water, sewage, dirt, debris,etc.
(Metagenomics)..(Important in ecology),
 As most viruses are too small to be seen by a light microscope, sequencing is one of the main tools in
virology to identify and study the virus.
 There are more than 2.3 million unique viral sequences in GenBank.

25. Conclusion
Organism Type Genome size Organization
Year of
completion
Caenorhabditis
elegans Nematode 100 Mb
Washington University and the Sanger
Institute
1998
Drosophila
melanogaster
Fruit fly 165 Mb
Celera, UC Berkeley, Baylor College of
Medicine, European DGP
2000
Anopheles gambiae
Mosquito 278 Mb Celera Genomics and Genoscope 2002
Takifugu rubripes Puffer fish 390 Mb
International Fugu Genome
Consortium
2002
Homo sapiens Human 3.2 Gb
Human Genome Project Consortium
and Celera Genomics 2006