all about dna sequencing

2. DNA Sequencing
Presented By:
Ayaz Ahmed
Muzammil Khalid
Aasim Ali
Presented To:
Dr. Yahya Noori

3. Objectives
 DNA Sequencing
 History of sequencing
 Purpose of it
 Types of DNA Sequencing
 Application of DNA Sequencing
 Future Perspective

4. DNA Sequencing
 Determining the order
of bases in a section
of DNA.
 To analyze gene
structure and its
relation to gene
expression as well as
protein conformation

5. History
 The first DNA fragment to be sequenced belonged to
a small virus called T4 bacteriophage that specifically
infects Escherichia coli bacteria. An important gene in
this organism codes for the enzyme, lysozyme. The
amino acid sequence of this enzyme had been
elucidated earlier through sequential digestion with
the enzyme trypsin, and the DNA sequence was
identified later.
 In the mid-1970s, Frederick Sanger improved this
initial method by using a plus-minus system for
running a sequencing reaction. In this modified
method, DNA polymerization initially occurred using
radiolabeled nucleotides.

7. History contn…
 After that, a short two-second pulse of polymerization was
done by adding or omitting a single nucleotide in each
reaction mixture. This created a set of eight reactions to give
a definitive picture of the nucleotide sequence within a DNA
molecule. The completed reactions were run on a
polyacrylamide gel for analysis. In this manner, the first
complete genome was sequenced, that of bacteriophage
ϕX174.
 Maxam and Gilbert further modified this method by busing
radiolabeled DNA and chemicals (such as hydrazine) that
would selectively induce the DNA molecule to break at certain
bases. Once again, the results of this chemical digestion
were analyzed on polyacrylamide gels.

9. Purpose of DNA Sequencing
 Deciphering “code of life”
 Detecting mutations
 Typing microorganisms
 Identifying human halotypes
 Designating polymorphisms

10. Types of DNA Sequencing
1-First Generation Sequencing
 Maxam & Gilbert (chemical degradation method)
Sanger Method (chain termination/Dideoxy method)
2-Genome Sequencing method
Shotgun sequencing
Clone contig approach
3-Second Generation Sequencing
Pyrosequencing
Illumina sequencing
Solid sequencing
Nanopore sequencing

11. Maxam & Gilbert
 A. M. Maxam and W.Gilbert-
1977
• The sequence of a double-
stranded or single-stranded
DNA molecule is determined by
treatment with chemicals that
cut the molecule at specific
nucleotide positions.

14. Sanger Method
 Most common approach
used for DNA
sequencing .
• Invented by Frederick
Sanger – 1977
• Nobel prize – 1980
• Also termed as Chain
Termination or Dideoxy
method

15. Sanger
The chain termination reaction
• Dideoxynucleotide triphosphates (ddNTPs) chain
terminators
having an H on the 3’C of the ribose sugar (normally OH
found in
dNTPs)
• ssDNA addition of dNTPs elongation
• ssDNA addition of ddNTPs elongation stops

16. Sanger method
 This method uses dideoxynucleotide triphosphates
(ddNTPs) chain terminators : which have an H on the 3’
carbon of the ribose sugar instead of the normal OH
found in deoxynucleotide triphosphates (dNTPs).
 Therefore in a synthesis reaction, if a dideoxynucleotide
is added instead of the normal deoxynucleotide, the
synthesis stops at that point because the 3’OH
necessary for the addition of the next nucleotide is
absent.

18. Principle of Sanger
 The sequence of a single-stranded DNA
molecule is determined by enzymatic synthesis
of complementary polynucleotide chains.
 These chains terminating at specific nucleotide
positions.
 Separate by gel electrophoresis Read DNA
sequence

29. Application of sequencing
 Forensics: to help identify
individuals because each
individual has a different genetic
sequence
 Medicine: can be used to help
detect the genes which are
linked to various genetic
disorders such as muscular
dystrophy.
 Diagnostic: for diagnosing
mutagenic disorders.
 Agriculture: The mapping and
sequencing of a genome of
microorganisms has helped to
make them useful for crops and
food plants.

30. Advantages Disadvantage
 Improved diagnosis of disease Whole genome can’t be
sequenced
at once.
 Bio pesticides Very Slow and time
consuming
 Identifying crime suspects

31. Future of DNA Sequencing
 Projects might focus on researching:
 The links to develop lifestyle
 Genomic and cardiovascular disease
 Early detections of cancer

32. NGS as Public Health tool
 ~0.25% of US women (375,000) carry a mutation in BRCA1/2
– At very high risk of breast and ovarian cancer
• 85% lifetime breast cancer risk
• 25-50% lifetime ovarian cancer cancer
 Knowledge of risk allows prevention
– Currently we only can identify such women once several family
members have developed cancer
 NGS allows population screening for high risk preventable disorders
– Cancer predisposition, cardiac disease, etc.
– ~1-2% of population carry such mutations
• 3-6 million individuals in the US with preventable disorders if
identified
 Doctors Urge Women To Test For Breast Cancer Gene After Jolie’s
Mastectomy

33. Challenges to harnessing NGS
in clinical medicine & public
health
 Accuracy
– 99.99% accuracy x 3 billion nucleotides
– = 300,000 errors per patient
 Interpretation of the variants we find
 Storage and access in the medical record
 Education of patients and public
 Issues of consent and reporting
 Education of providers