DNA Sequencing

2nd year MLT

2016
Quick Reminder

Adenine
Guanine Purine

Thymine
Cytosine Pyrimidine
Uracil

Hydrogen Bonding
A T
C G
 DNA Sequencing
• Definition:
The process of determining the precise
order of nucleotides in a DNA molecule
• Application:
DNA sequencing is applied in many
biological studies including; forensic Fred Sanger
researches, medical diagnostics,
biotechnology, and virology.
• Founders:
Fred Sanger and Walter Gilbert – Nobel
prize in chemistry 1980 Walter Gilbert
 Sequencing Methods
• Two basic methods:
1. Chemical cleavage method (Maxam and
Gilbert method, 1976-77)
• the first widely adopted method for DNA sequencing.
• partial chemical modification of DNA and subsequent
cleavage of the DNA backbone at sites adjacent to the
modified nucleotides.
• no longer in widespread use.
2. Chain terminating inhibitors (Sanger method,
1977)
• based on the selective incorporation of chain-terminating
dideoxynucleotides by DNA polymerase during DNA
elongation step.
 Chemical cleavage
method
• Separation of two DNA
strands.
• Labeling 5’ end of DNA
with 32P.
• Division of the mixture
into four tubes, each
treated with different
regent to cleave either G
only, C only, Purine
(A+G), or Pyrimidine
(T+C).
Electrophoresis
Reading

• Electrophoresis in four
separate lanes of the gel.
• Radiography the gel and
determining the
sequence.
Classical Sanger Sequencing Method
Automated Sanger Sequencing
Method
 Automated Sanger Sequencing
Method
• Similar to the manual (classical) method.
• Reaction carried out in ONE tube and all
possible products are produced in one
reaction.
• The various reaction products separate
according to size on gel electrophoresis
(capillary gel)
• Each of the ddNTPs is color coded and laser
scanner excites the fluorescent to be detected
by a detector which analyze the emitted light
and plot it as a colored peak.
Printout of an Automated Sanger
Sequencing Method
Normal (Wild-Type)

SNP/Point Mutation
Using only one
primer either
Forward or Reverse…
why?
Next Generation Sequencing (NGS)
• also known as high-throughput sequencing
• Next generation sequencing applies to
genome sequencing
Ion Torrent, Illumina, or SOLiD
• Two types:
• Whole Genome Sequencing (WGS)
The most comprehensive method of interrogating the 3.2
billion bases of the human genome
• Whole Exome Sequencing (WES)
Exon/Exome?!!
<2% of the genome is Exome
 Why WES not WGS?

• Protein coding genes (exome) <2% of the

genome but contain 85% of disease causing
mutations.
• Cheaper than sequencing entire genome.
• WGS data storage and analysis are challenging
• After the quick development of sequencing,
many mutation and gene variation (SNP)
databases are now available to public:
COSMIC: Catalogue Of Somatic Mutations In
Cancer (for majority types of cancer)
HGMD: The Human Gene Mutation Database
(for human inherited disease)
Leiden Muscular Dystrophy data pages:
muscular dystrophy mutation databases
Breast Cancer Mutation Data Base (BIC)
Hb variant database: A Database of Human
Hemoglobin Variants and Thalassemias