Presented by,
Raihanathus Sahdhiyya A,
I M.Sc. Microbiology
What is DNA sequencing?
Process of determining the sequence of nucleotides
Adenine
Thymine
Cytosine
Guanine
in a piece of DNA.
Sanger sequencing
Fredrick Sanger with his colleagues developed this method of
sequencing in the year 1977, for which he was awarded Nobel in
1980
It is also known as Chain termination method, Dideoxy sequencing
or Enzymatic method
It was the most widely used DNA sequencing method for almost 40
years, bringing successful completion of the Human Genome Project
(HGP) in 2003
Sanger sequencing method is based on the chain termination by the
use of Dideoxynucleotides (ddNTPs)
What is Dideoxynucleotide ?
(ddNTPs)
A dideoxynucleotide (ddNTP) is an artificial molecule that lacks a
hydroxyl group at the 3' carbon of the ribose sugar
Sanger sequencing
Sanger sequencing process
1. DENATURE & CLONE
The first step is to fragment the DNA by applying heat and
clone the fragments into vectors
2. PRIMER ATTACHMENT
• The second step is to anneal a synthetic oligonucleotide
• The oligonucleotide acts as a binding site for a primer and
provides a 3' hydroxyl group, which is necessary to initiate
DNA synthesis
• In order to recognize the sequence and identify precisely the
first nucleotide of the target DNA, the primer is usually
positioned 10 to 20 nucleotides away from the target DNA.
3. ADDITION OF dNTP s AND ddNTPs
• Four different reaction vials are made, each with the four
standard dNTP's, and DNA polymerases
•The difference among the vials are the type of ddNTPs. Each
vial will have 1 ddNTP per 100 dNTP
(contd.)
• After DNA synthesis occurs, each reaction vial will have a
unique set of single-stranded DNA molecules of varying
lengths. However, all DNA molecules will have the same
primer sequence at its 5' end
•The resulting DNA fragments are then denatured by heat
since base-paired loops of ssDNA may cause difficulty in
resolving bands when running a gel. Additionally, formamide
may also be added to prevent base pairing.
4. SEQUENCING BY GEL ELECTROPHORESIS
• As the sequences vary in size, they have to be lined up
according to the size to determine the sequence
•Radiography
Here, the ddNTPs would
have to be radioactively or
fluorescently labeled beforehand
for automated sequencing
machines. The DNA strands are
then separated using gel
electrophoresis, then read from
top to bottom (3' to 5') to obtain
the sequence.
(contd.)
• Dye-terminating sequencing
Each ddNTP is fluorescently labeled to use dye-terminating
sequencing. This causes each of the four ddNTPs to emit light at
different wavelengths. Here, capillary electrophoresis is carried
out, with a single lane to capture the nucleotide sequence.
ADVANTAGES
Not as toxic and less radioactivity than Maxam and Gilbert
method
Easier to automate - Leroy Hood and coworkers used
fluorescently labeled ddNTPs and primers for the first high-
throughput DNA sequencing machine. This lowered the cost from
$100 million to $10,000 USD in 2011
Highly accurate long sequence reads of about 700 base pairs
Easier to get started. The kits that are commercially available
contain reagents necessary for sequencing - pre-aliquoted and
ready to use.
DISADVANTAGES
Poor quality in the first 15-40 bases of the sequence. This is due
to primer binding and deteriorating quality of sequencing traces
after 700-900 bases
Time consuming, especially due to requirement for
electrophoretic separation of fragments
DNA fragments cloned before sequencing - read may include
parts of the cloning vector
Only short 300-1000 nucleotides long DNA fragments in a
single reaction. Problem with reading strands longer is the
insufficient power of separation for resolving large DNA
fragments that differ in length by only one nucleotide.
