Reminder:
All molecular techniques are based
on the chemical “personality” (or chemical
properties) of the DNA molecule (or nucleic acids)
DNA Sequencing
DNA sequencing
✓ Determination of nucleotide sequence
✓ the determination of the precise sequence of nucleotides in
a sample of DNA
✓ Two similar methods:
1. Maxam and Gilbert method
2. Sanger method
✓ They depend on the production of a mixture of oligonucleotides
labeled either radioactively or fluorescein, with one common end and
differing in length by a single nucleotide at the other end
✓ This mixture of oligonucleotides is separated by high resolution
electrophoresis on polyacrilamide gels and the position of the bands
determined
The Maxam-Gilbert
Technique
◼ Principle:
Chemical Degradation of Purines
• Purines (A, G) damaged by
dimethylsulfate
• Methylation of base
• Heat releases base
• Alkali cleaves G
• Dilute acid cleave A>G
Maxam-Gilbert
Technique
• Pyrimidines (C, T) are
damaged by hydrazine
• Piperidine cleaves the
backbone
• 2 M NaCl inhibits the
reaction with T
Maxam and Gilbert Method
✓ Chemical degradation of purified fragments (chemical
degradation)
✓ The single stranded DNA fragment to be sequenced is end-
labeled by treatment with alkaline phosphatase to remove the
5’phosphate
✓ It is then followed by reaction with P-labeled ATP in the
presence of polynucleotide kinase, which attaches P labeled to
the 5’terminal
✓ The labeled DNA fragment is then divided into four aliquots,
each of which is treated with a reagent which modifies a
specific base
Maxam and Gilbert Method
1. Aliquot A + dimethyl sulphate, which methylates guanine
residue
2. Aliquot B + formic acid, which modifies adenine and
guanine residues
3. Aliquot C + Hydrazine, which modifies thymine + cytosine
residues
4. 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
Maxam-Gilbert
sequencing - modifications
Maxam-Gilbert sequencing:
Summary
Advantages/disadvantages
Maxam-Gilbert sequencing
✓ Requires lots of purified DNA, and many intermediate
purification steps
✓ Relatively short readings
✓ Automation not available (sequencers)
✓ Remaining use for ‘footprinting’ (partial protection against
DNA modification when proteins bind to specific regions, and
that produce ‘holes’ in the sequence ladder)
In contrast, the Sanger sequencing methodology requires
little if any DNA purification, no restriction digests,
and no labeling of the DNA sequencing template
Sanger
✓ Fred Sanger, 1958
• Was originally a protein
chemist
• Made his first mark in
sequencing proteins
• Made his second mark
in sequencing RNA
✓ 1980 dideoxy
sequencing
Original Sanger Method
✓ Random incorporation of a dideoxynucleoside
triphosphate into a growing strand of DNA
✓ Requires DNA polymerase I
✓ Requires a cloning vector with initial primer (M13,
high yield bacteriophage, modified by adding: beta-
galactosidase screening, polylinker)
✓ Uses 32P-deoxynucleoside triphosphates
Sanger Method
✓ in-vitro DNA synthesis using ‘terminators’, use of dideoxi-
nucleotides that do not permit chain elongation after their
integration
✓ DNA synthesis using deoxy- and dideoxynucleotides that
results in termination of synthesis at specific nucleotides
✓ Requires a primer, DNA polymerase, a template, a
mixture of nucleotides, and detection system
Sanger Method
✓ Incorporation of di-deoxynucleotides into growing strand
terminates synthesis
✓ Synthesized strand sizes are determined for each di-
deoxynucleotide by using gel or capillary electrophoresis
✓ Enzymatic methods
Dideoxynucleotide
5’
PPP O CH2 BASE
O
3’
no hydroxyl group at 3’ end
prevents strand extension
The principles
✓ Partial copies of DNA fragments made with DNA
polymerase
✓ Collection of DNA fragments that terminate with
A,C,G or T using ddNTP
✓ Separate by gel electrophoresis
✓ Read DNA sequence
3’ CCGTAC 5’
primer 5’ 3’
dNTP
ddATP ddTTP ddCTP ddGTP
GGCA GGCAT GGC G
GG
GGCATG
A T C G
Chain Terminator Basics
Target
Template-Primer
TGCA
ddA ddC
Extend ddG
ddA ddT
Labeled Terminators
AddC
dN : ddN
AC ddG 100 : 1
ACG ddT
Electrophoresis
Sanger Method Sequencing Gel
Sequencing of DNA by the Sanger method
Comparison
◼ Sanger Method ◼ Maxam Gilbert Method
• Enzymatic • Chemical
• Requires DNA synthesis • Requires DNA
• Termination of chain • Requires long stretches of
elongation DNA
• Breaks DNA at different
nucleotides
ASSIGNMENT
◼ Sequence the DNA using Sanger method
3’-CGAATCCGGATCGATATCGG5’
