Download - Dna Acid Sequencing
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DNA SEQUENCING
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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
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TECHNIQUES
Maxam Gilbert method
Sanger’s method
Other techniques
- Automated Sequencing
- Pyro sequencing
- Microarrays
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Maxam-Gilbert method
• Allan Maxam – Walter Gilbert
• 1976
• Harward physist
• First developed sequencing method
• Chemical method
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Requirement
DNA sample
NaOH
Chemicals
PAGE
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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
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Chemical Reagents Used
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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.
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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
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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
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Sangers method
• 1980
• Fred Sanger – protein chemist
• Nobel laureate
• Also,
Dideoxynucleotide method
Chain termination method
Enzymatic method
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Dideoxy dNTP
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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)
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Steps involved
- Template
- Primer (radio labeled)
- Polymerase 1
- Extension chemistry
- Termination (by ddNTP)
- Separation
- Detection
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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).
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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
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Sample Output
1 lane
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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).
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Thank you….,