dna acid sequencing
TRANSCRIPT
DNA SEQUENCING
OBJECTIVE
• To determine the nucleotide sequences present in the given DNA sample.
WHY??
To study the genomic organization of a organism
To identify the restriction sites in plasmids
Cloning
TECHNIQUES
Maxam Gilbert method
Sanger’s method
Other techniques
- Automated Sequencing
- Pyro sequencing
- Microarrays
Maxam-Gilbert method
• Allan Maxam – Walter Gilbert
• 1976
• Harward physist
• First developed sequencing method
• Chemical method
Requirement
DNA sample
NaOH
Chemicals
PAGE
Chemical Degradation of Purines
– Purines ( G) damaged by dimethylsulfate
– Methylation of base
– Heat releases base
– Alkali cleaves G
– Formic acid – modifies A,G
Chemical Degradation of Pyrimidines
– Pyrimidines (C, T) are damaged by hydrazine
– Piperidine cleaves the backbone
– 2 M NaCl inhibits the reaction with T
Chemical Reagents Used
Steps Involved
Denature the ds DNA with NaOH
5’ end of the single stranded DNA fragment to be sequenced is P-labelled
The labeled DNA fragment is then divided into four aliquots,
1. Aliquot A + dimethyl sulphate, which methylates guanine residue2. Aliquot B + formic acid, which modifies adenine and guanine residues3. Aliquot C + Hydrazine, which modifies thymine + cytosine residues4. Aliquot D + Hydrazine + 5 mol/l NaCl, which makes the reaction specific for cytosine
The four are incubated with piperidine which cleaves the sugar phosphate backbone of DNA next to the residue that has been modified.
Advantages
Homopolymeric DNA runs are sequenced as efficiently as heterogeneous
DNA sequences,
Used to analyze DNA-protein interactions (i.e., footprinting)
Used to analyze nucleic acid structure and epigenetic modifications to
DNA
Limitation
However, despite all improvements, these limitations remain:
(1) Gel electrophoresis is limited to 700-900 bp, with 400-500 bp more commonly attained
(2) The first 15-40 bp are often difficult to interpret
(3) Requires lots of purified DNA, and many intermediate purification steps
Disadvantages
This method is not commonly used today because:
(a) it requires extensive use of hazardous chemicals,
(b) it has a relatively complex set-up/technical complexity,
(c) it is difficult to "scale-up", and cannot be used to analyze more than 500 base pairs
Sangers method
• 1980
• Fred Sanger – protein chemist
• Nobel laureate
• Also,
Dideoxynucleotide method
Chain termination method
Enzymatic method
Dideoxy dNTP
Requirement
1. Single stranded template or target DNA
2. Sequence-specific primer
3. DNA polymerase
4. All 4 dNTP’s (dATP, dGTP, dCTP, dTTP)
5. All 4 ddNTP’s (ddATP, ddGTP, ddCTP, ddTTP)
Steps involved
- Template
- Primer (radio labeled)
- Polymerase 1
- Extension chemistry
- Termination (by ddNTP)
- Separation
- Detection
Problem
A cloned fragment of DNA was sequenced by using the dideoxy
method. A part of the autoradiogram of the sequencing gel is
represented here.
• Deduce the nucleotide sequence of the DNA nucleotide chain
synthesized from the primer. Label the 5′ and 3′ ends.
• Deduce the nucleotide sequence of the DNA nucleotide chain used
as the template strand. Label the 5′ and 3′ ends.
• Write the nucleotide sequence of the DNA double helix (label the 5′
and 3′ ends).
Comparison
• Sanger Method
– Enzymatic
– Requires DNA synthesis
– DNA is labeled
– Termination of chain
elongation
– Large base sequence
• Maxam Gilbert Method
– Chemical
– Requires DNA
– Primer is labeled
– Breaks DNA at different
nucleotides
– Small base sequence
Sample Output
1 lane
Summary
• Genetic information is stored in the order or sequence of nucleotides
in DNA.
• Chain termination sequencing is the standard method for the
determination of nucleotide sequence.
• Dideoxy-chain termination sequencing has been facilitated by the
the use of fluorescent dye detection.
• Alternative methods are used for special applications, such as
pyrosequencing (for resequencing and polymorphism detection) or
bisulfate sequencing (to analyze methylated DNA).
Thank you….,