Download Western Blot - Faperta UGM

Reminder:
All molecular techniques are based
on the chemical “personality” (or chemical
properties) of the DNA molecule (or nucleic acids)
Studies of cell
-Fractionation
-Purification/ Identification
-Structure/ Function
Organelle level
Cell fractionation
-Nucleus
-Mitochondria
-Ceell membrane
-Cytosol
Cellular level
Microscope
Molecular level: Macromolecules
Proteins
Carbohydrates
Lipids
Nucleic acids
Atomic level
C, H, O, N, S, P
Negatively-charged phosphate-sugar backbone
Various lengths
-
-
-
Specificity of nucleotides
Hydrogen bonds
CONTENTS
Enzymes
Electrophoresis
Blotting and Hybridization
Polymerase Chain Reaction
DNA Sequences
Enzymes


Large molecules made of various amino acids
Act as catalysts to speed up reactions w/out being
destroyed
•
•
•
•
•
Increase the rate of reaction
Highly specific
Lowers energy of activation level
Activity lost if denatured
May contain cofactors such as metal ions or organic
(vitamins)
Name of Enzymes



End in –ase
Identifies a reacting substance
1. sucrase – reacts sucrose
2. lipase - reacts lipid
Describes function of enzyme
1. oxidase – catalyzes oxidation
2. hydrolase – catalyzes hydrolysis
6
Classification of Enzymes
Class
 Oxidoreductoases
 Transferases
 Hydrolases
 Lyases


Isomerases
Ligases
Reactions catalyzed
oxidation-reduction
transfer group of atoms
hydrolysis
add/remove atoms to/from a
double bond
rearrange atoms
combine molecules
Enzyme Action:
Lock and Key Model






An enzyme binds a substrate in a region called the active
site
Only certain substrates can fit the active site
Amino acid R groups in the active site help substrate
bind
Enzyme-substrate complex forms
Substrate reacts to form product
Product is released
8
Lock and Key Model
P
+
S
+
S
P
E
+ S
ES complex
E
+
P
Enzymes use in
Molecular Genetics
1. Restriction endonucleases/enzymes
2. Ligase
3. DNA polymerase
Restriction Enzymes
Molecular scissors which isolated from bacteria where they are used
as Bacterial defense against viruses
Molecular scalpels to cut DNA in a precise and predictable manner
Enzyme produced by bacteria that typically recognize specific 4-8
base pair sequences called restriction sites, and then cleave both
DNA strands at this site
A class of endo-nucleases that cleavage DNA after recognizing a
specific sequence
Members of the class of nucleases
Nuclease
Breaking the phosphodiester bonds that link adjacent
nucleotides in DNA and RNA molecules
 Endonuclease
Cleave nucleic acids at internal position
 Exonuclease
Progressively digest from the ends of the nucleic acid
molecules
Endonuclease
Type
Characteristics
I

II
III
Have both restriction and modification activity
 Cut at sites 1000 nucleotides or more away from recognition site
 ATP is required
 It has only restriction site activity
 Its cut is predictable and consistent manner at a site within or
adjacent to restriction site
 It require only magnesium ion as cofactor
Have both restriction and modification activity
Cut at sites closed to recognition site
ATP is required
Restriction Enzymes
 There are already more than 1200 type II enzymes isolated from
prokaryotic organism
 They recognize more than 130 different nucleotide sequence
 They scan a DNA molecule, stopping only when it recognizes a specific
sequence of nucleotides that are composed of symetrical, palindromic
sequence
Palindromic sequence:
The sequence read forward on one DNA strand is identical to the
sequence read in the opposite direction on the complementary strand
 To Avoid confusion, restriction endo-nucleases are named according to
the following nomenclature
Nomenclature
 The first letter is the initial letter of the genus name of the
organism from which the enzyme is isolated
 The second and third letters are usually the initial letters of the
organisms species name. It is written in italic
 A fourth letter, if any, indicates a particular strain organism
 Originally, roman numerals were meant to indicate the order in
which enzymes, isolated from the same organisms and strain,
are eluted from a chromatography column. More often, the
roman numerals indicate the order of discovery
Nomenclature
EcoRI
E : Genus Escherichia
co: Species coli
R : Strain RY13
I : First endonuclease isolated
BamHI
B : Genus Bacillus
am: species amyloliquefaciens
H : Strain H
I : First endonuclease isolated
HindIII
H : Genus Haemophilus
in : species influenzae
d : strain Rd
III : Third endonuclease isolated
Specificity
Enzyme
Source
Sequence
End
BamHI
Bacillus amyloliquefaciens H
GGATCC
Sticky
BglII
Bacillus globigii
AGATCT
Sticky
EcoRI
Escherichia coli RY13
GAATTC
Sticky
EcoRII
Escherichia coli R245
CCTGG
Sticky
HaeIII
Haemophilus aegyptius
GGCC
Blunt
HindII
Haemophilus influenzae Rd
GTPyPuAC
Blunt
HindIII
Haemophilus influenzae Rd
AAGCTT
Sticky
HpaII
Haemophilus parainfluenzae
CCGG
Sticky
NotI
Nocardia otitidis-caviarum
GCGGCCGC Sticky
PstI
Providencia stuartii 164
CTGCAG
Sticky
Restriction Product
Restriction enzymes
Restriction enzymes can be grouped by:
number of nucleotides recognized (4, 6,8 base-cutters most
common)
kind of ends produced (5’ or 3’ overhang
(cohesive=sticky), blunt=flush)
degenerate or specific sequences
whether cleavage occurs within the recognition
sequence
A restriction enzyme (EcoRI)
1. 6-base cutter
2. Specific palindromic
sequence
(5’GAATTC)
3. Cuts within the
recognition
sequence (type II
enzyme)
4. produces a 5’
overhang (sticky end)
Ligase
Any of a class of enzymes that act as
catalysts in chemical reactions in
which molecules are linked together,
as in the synthesis and repair of
DNA or in the formation of
recombinant DNA
Any of a class of enzymes that
catalyze the linkage of two molecules,
generally utilizing ATP as the energy
donor (synthetase).
Function of DNA ligase
The enzyme, DNA ligase,
repairs the millions of
DNA breaks generated
during the normal course
of a cell's life, for example,
linking together the
abundant DNA fragments
formed during replication
of the genetic material in
dividing cells.
Ligase
EC 6
Ligases
EC 6.1
Forming carbon—oxygen bonds
EC 6.2
Forming carbon—sulfur bonds
EC 6.3
Forming carbon—nitrogen bonds
EC 6.4
Forming carbon—carbon bonds
EC 6.5
Forming phosphoric ester bonds
EC 6.6
Forming nitrogen—metal bonds
DNA Ligase Mechanism
DNA Ligase Mechanism
Human DNA Ligase
Human DNA ligase III gene encodes
both nuclear and mitochondrial enzymes.
DNA ligase plays a central role in DNA
replication, recombination, and DNA
repair.
DNA Polymerase



an enzyme that is template
based and has both 5’->3' DNA
polymerase activity and 3’->5'
exonuclease activity.
highly processive, meaning it
synthesizes long stretches of
DNA without dissociating from
the DNA template.
an open right hand, composed
of a thumb domain that binds
to thioredoxin, a finger
domain in which catalytic
activity resides, a palm
domain that cradles the DNA,
and a terminal exonuclease
domain
Three main features of the DNA
synthesis reaction
1. DNA polymerase I catalyzes formation of phosphodiester
bond between 3’-OH of the deoxyribose (on the last
nucleotide) and he 5’-phosphate of the dNTP.
Energy for this reaction is derived from the release of two of
the three phosphates of the dNTP.
2. DNA polymerase “finds” the correct complementary dNTP
at each step in the lengthening process.
• rate ≤ 800 dNTPs/second
• low error rate
3. Direction of synthesis is 5’ to 3’
DNA elongation
DNA elongation
Types of DNA polymerase
Polymerase Polymerization Exonuclease Exonuclease
I
II
III
(5’-3’)
Yes
Yes
Yes
(3’-5’)
Yes
Yes
Yes
(5’-3’)
Yes
No
No
#Copies
400
?
10-20
•3’ to 5’ exonuclease activity : ability to remove nucleotides from the
3’ end of the chain
•Important proofreading ability
•Without proofreading error rate (mutation rate) is 1 x 10-6
•With proofreading error rate is 1 x 10-9 (1000-fold decrease)
•5’ to 3’ exonuclease activity : the ability in DNA replication & repair.
Eukaryotic enzymes
Five common DNA polymerases from mammals.
1. Polymerase  (alpha): nuclear, DNA replication, no
proofreading
2. Polymerase  (beta): nuclear, DNA repair, no proofreading
3. Polymerase  (gamma): mitochondria, DNA repl., proofreading
4. Polymerase  (delta): nuclear, DNA replication, proofreading
5. Polymerase  (epsilon): nuclear, DNA repair (?), proofreading
•
•
•
•
Different polymerases for the nucleus and mtDNA
Some polymerases proofread; others do not.
Some polymerases used for replication; others for repair.
Polymerases vary by species.
In this illustration, DNA ligase (in color) encircles the
DNA double helix.
Researchers investigating an
important DNA-repair enzyme
now have a better picture of the
final steps of a process that
glues together, or ligates, the
ends of DNA strands to restore
the double helix.
The enzyme, DNA ligase, repairs
the millions of DNA breaks
generated during the normal
course of a cell's life, for
example, linking together the
abundant DNA fragments formed
during replication of the genetic
material in dividing cells.
GEL ELECTROPHORESIS
The motion of disperse charged particle relatives to a fluid
under the influence of a spatially uniform electric field
First observed by Reuss, 1807
1. For separating disperse charged
biological molecule of any
size/length
2. Uses electricity
3. Uses a matrix
4. Uses buffer solution
Electrophoresis
Factors affecting the mobility of molecules:
1. Molecular factors
• Charge
• Size
• Shape
-
2. Environment factors
• Electric field strength
• Matrix (pore: sieving effect)
• Running buffer
+
Electrophoresis
Types of matrix (supporting media)
 Paper
 Agarose
1. purified large MW polysaccharide (from agar)
2. very open (large pore) gel
3. used frequently for large DNA molecules
 Acrylamide
1. a white odorless crystalline solid chemical compound
2. soluble in water, ethanol, ether, chloroform
3. used to synthesize poly-acrylamide which find many uses as
water soluble thickeners
 Starch
 Cellulose acetate
DNA Agarose Gel
An analytical technique used to separate DNA by size
1. Electric field induces DNA to
migrate toward the anode due
to the net negative charge of
the sugar phosphate backbone
of the DNA
2. Longer molecules migrate
more slowly
3. Visualized using a
fluorescence dye special for
DNA such as ethidium
bromide
Polyacrylamide Gels




acrylamide polymer
very stable gel
can be made at a wide variety of concentrations
large variety of pore sizes (powerful sieving effect)
SDS-Polyacrylamide Gel
Electrophoresis (SDS-PAGE)
 Sodium Dodecyl Sulfate = Sodium Lauryl
Sulfate: CH3(CH2)11SO3- Na+
 Amphipathic molecule
 Strong detergent to denature proteins
 Binding ratio: 1.4 g SDS/g protein
 Charge and shape normalization
Isoelectric Focusing
Electrophoresis (IFE)
- Separate molecules
according to their
isoelectric point (pI)
- At isoelectric point (pI)
molecule has no charge
(q=0), hence molecule
ceases
- pH gradient medium
2-dimensional Gel
Electrophoresis
- First dimension is IFE
(separated by charge)
- Second dimension is SDSPAGE (separated by size)
- So called 2D-PAGE
- High throughput
electrophoresis, high resolution
2-dimensional Gel Electrophoresis
Spot coordination
 pH
 MW
Blotting and
Hybridization
Blotting
Transfer the DNA from the gel to a solid support
Transferring of DNA, RNA, Protein to an immobilizing
binding matrix such as nitrocellulose paper or nylon
Northern blot
(RNA)
Eastern blot
(???)
Western blot
(Protein)
Southern blot
(DNA)
Blotting
• Two methods :
– Capillary transfer
– Electrophoretic transfer
SOUTHERN BLOTTING
The technique was developed by E.M. Southern in
1975.
 The Southern blot is used to detect the presence of a
particular piece of DNA in a sample.
 The DNA detected can be a single gene, or it can be
part of a larger piece of DNA such as a viral genome
 The key to this method is hybridization.
 Hybridization-process of forming a double-stranded
DNA molecule between a single-stranded DNA probe
and a single-stranded target patient DNA.

SOUTHERN BLOTTING
There are 2 important features of hybridization:
• The reactions are specific
The probes will only bind to targets with a
complementary sequence.
• The probe can find one molecule of target in a
mixture of millions of related but noncomplementary molecules.
Southerns Blotting
(DNA Blotting)




DNA fragments created by restriction
digestion are separated on an agarose gel
Separated fragments are denatured and
transferred to a membrane (blot) by blotting
Probe is hybridized to complementary
sequences on the blot and excess probe is
washed away
Location of probe is determined by detection
method (e.g., film, fluorometer)
Southern blotting
Some Applications of DNA Blots


Map restrictions sites near a particular locus for
gene isolation or allele analysis (e.g., RFLP
restriction fragment length polymorphism)
 Identity of closely related genes
Confirmation of gene transfer or gene disruption
 Detection of foreign DNA
RNA Blotting (Northern)
Use DNA to prove RNA
RNA is separated by size on a denaturing agarose gel
and then transferred onto a membrane (blot)
 Probe is hybridized to complementary sequences on the
blot and excess probe is washed away
 Location of probe is determined by detection method
(e.g., film, fluorometer)

RNA Blotting (Northern)
RNA Mixture
RNA
RNA
RNA Blotting (Northern)
Advantage:
- Very sensitive
- Blots are reusable
- Technical protocol is relatively simple
- Can detect mRNA splice variants
Disadvantage:
- Use of radioactivity
(although non-radioactive techniques are available)
- Laborious if many genes need to be tested
- Assay is time-consuming
Applications of RNA Blots
Detect the expression level and transcript size of a
specific gene in a specific tissue or at a specific
time. Sometimes mutations do not affect coding
regions but transcriptional regulatory sequences
(e.g., UAS/URS, promoter, splice sites, copy
number, transcript stability)
Western Blot
 Protein blotting
 Highly specific qualitative test
 Can determine if above or below threshold
 Typically used for research
 Use denaturing SDS-PAGE
 Solubilizes, removes aggregates & adventitious proteins are
eliminated
Components of the gel are then transferred to a solid
support or transfer membrane
weight
Transfer
membrane
Paper towel
Paper towel
Wet filter paper
Western Blot
Hybridization
Pairing of complementary DNA and/or RNA and/or protein
Hybridization






It can be DNA:DNA, DNA:RNA, or RNA:RNA
(RNA is easily degraded)
It depended on the extent of complementation
It depended on temperature, salt concentration, and
solvents
Small changes in the above factors can be used to
discriminate between different sequences (e.g. small
mutations can be detected)
Probes can be labeled with radioactivity, fluorescent
dyes, enzymes.
Probes can be isolated or synthesized sequences
In-situ hybridisation
Hybridization which is performed by denaturing the DNA of
cell squash on a microscope slide so that reaction is possible with
an added of probe


Chromosome in-situ hybridisation
 DNA probe detects sequences in chromosomes
 Map gene sequences
Tissue in-situ hybridisation
 RNA probe detects sequences in cells and tissues
 Identify sites of gene expression
 Analyse tissue distribution of expression
Oligonucleotide probes



Single stranded DNA (usually 15-40 bp)
Degenerate oligo-nucleotide probes can be used to
identify genes encoding characterized proteins
• Use amino acid sequence to predict possible DNA
sequences
• Hybridize with a combination of probes
• TT(T/C) - TGG - ATG - GA(T/C) - TG(T/C) could be used for FWMDC amino acid sequence
Can specifically detect single nucleotide changes
Detection of Probes




Probes can be labeled with radioactivity, fluorescent
dyes, enzymes.
Radioactivity is often detected by X-ray film
(autoradiography)
Fluorescent dyes can be detected by fluorometers,
scanners
Enzymatic activities are often detected by the
production of dyes or light (x-ray film)
Polymerase Chain Reaction
(PCR)
Polymerase Chain Reaction
Powerful technique for amplifying DNA
Amplified DNA are then separated
by gel electrophoresis
PCR
 A simple rapid, sensitive and versatile in vitro method for selectively
amplifying defined sequences/regions of DNA/RNA from an initial
complex source of nucleic acid - generates sufficient for subsequent
analysis and/or manipulation
 Amplification of a small amount of DNA using specific DNA primers
(a common method of creating copies of specific fragments of DNA)
 DNA fragments are synthesized in vitro by repeated reactions of DNA
synthesis (It rapidly amplifies a single DNA molecule into many billions
of molecules)
 In one application of the technology, small samples of DNA, such as
those found in a strand of hair at a crime scene, can produce sufficient
copies to carry out forensic tests.
 Each cycle the amount of DNA doubles
Background on PCR
 The ability to generate identical high copy number DNAs made
possible in the 1970s by recombinant DNA technology (i.e.,
cloning).
 Cloning DNA is time consuming and expensive
 Probing libraries can be like hunting for a needle in a haystack.
 Requires only simple, inexpensive ingredients and a couple hours
 PCR, “discovered” in 1983 by Kary Mullis,
 Nobel Prize for Chemistry (1993).
 It can be performed by hand or in a machine called a thermal
cycler.
Three Steps

Separation
Double Stranded DNA is denatured by heat into single strands.
Short Primers for DNA replication are added to the mixture.

Priming
DNA polymerase catalyzes the production of complementary new
strands.

Copying
The process is repeated for each new strand created
All three steps are carried out in the same vial but at different
temperatures
Step 1: Separation


Combine Target Sequence, DNA primers template,
dNTPs, Taq Polymerase
Target Sequence
1. Usually fewer than 3000 bp
2. Identified by a specific pair of DNA primers- usually oligonucleotides that
are about 20 nucleotides

Heat to 95°C to separate strands (for 0.5-2 minutes)
• Longer times increase denaturation but decrease enzyme and template
Magnesium as a Cofactor
Mg stabilizes the reaction between:
• oligonucleotides and template DNA
• DNA Polymerase and template DNA
Heat
Denatures DNA by uncoiling the Double Helix strands.
Step 2: Priming
Decrease temperature by 15-25 °C
 Primers anneal to the end of the strand
 0.5-2 minutes
Shorter time increases specificity but decreases yield
Requires knowledge of the base sequences of the 3’ end



Selecting a Primer
Primer length
 Melting Temperature (Tm)
 Specificity
 Complementary Primer Sequences
G/C content and Polypyrimidine (T, C) or
polypurine (A, G) stretches
 3’-end Sequence
 Single-stranded DNA


Step 3: Polymerization



Since the Taq polymerase works best
at around 75 ° C (the temperature of
the hot springs where the bacterium
was discovered), the temperature of
the vial is raised to 72-75 °C
The DNA polymerase recognizes the
primer and makes a complementary
copy of the template which is now
single stranded.
Approximately 150 nucleotides/sec
Potential Problems with Taq
Lack of proof-reading of newly synthesized DNA.
 Potentially can include di-Nucleotriphosphates
(dNTPs) that are not complementary to the original
strand.
 Errors in coding result
Recently discovered thermostable DNA polymerases,
Tth and Pfu, are less efficient, yet highly accurate.


How PCR works
1. Begins with DNA containing a sequence to be amplified and a
pair of synthetic oligonucleotide primers that flank the sequence.
2. Next, denature the DNA at 94˚C.
3. Rapidly cool the DNA (37-65˚C) and anneal primers to
complementary s.s. sequences flanking the target DNA.
4. Extend primers at 70-75˚C using a heat-resistant DNA
polymerase (e.g., Taq polymerase derived from Thermus aquaticus).
5. Repeat the cycle of denaturing, annealing, and extension 20-45
times to produce 1 million (220) to 35 trillion copies (245) of the
target DNA.
6. Extend the primers at 70-75˚C once more to allow incomplete
extension products in the reaction mixture to extend completely.
7. Cool to 4˚C and store or use amplified PCR product for analysis.
Thermal cycler protocol Example
Step 1 7 min at 94˚C
Step 2 45 cycles of:
20 sec at 94˚C
20 sec at 64˚C
1 min at 72˚C
Step 3 7 min at 72˚C
Step 4 Infinite hold at 4˚C
Initial Denature
Denature
Anneal
Extension
Final Extension
Storage
The Polymerase Chain Reaction
PCR amplification
Each cycle the oligo-nucleotide primers bind
most all templates due to the high primer
concentration
The generation of mg quantities of DNA can be
achieved in ~30 cycles (~ 4 hrs)


OPTIMISING PCR
THE REACTION COMPONENTS
Starting nucleic acid - DNA/RNA
 Thermo-stable DNA polymerase
e.g. Taq polymerase
 Oligonucleotides (primer)
Design them well!
 Buffer
Tris-HCl (pH 7.6-8.0)
Mg2+
dNTPs (dATP, dCTP, dGTP, dTTP)
RAW MATERIAL
 Organims, Organ, Tissue, cells (hair root, callus, leaves, root, seed)
 Obtain the best starting material.
 Some can contain inhibitors of PCR, so they must be removed
e.g. Haem in blood
 Good quality genomic DNA if possible
 Empirically determine the amount to add
POLYMERASE
 Number of options available
Taq polymerase
Pfu polymerase
Tth polymerase
 How big is the product?
100bp
40-50kb
 What is end purpose of PCR?
1. Sequencing - mutation detection
-. Need high fidelity polymerase
-. integral 3’ ________ 5' proofreading exonuclease activity
2. Cloning
3. Marker development
PRIMER DESIGN
Length ~ 10-30 nucleotides (21 nucleotides for gene isolation)
Base composition:
50 - 60% GC rich, pairs should have equivalent Tms
Tm = [(number of A+T residues) x 2 °C] + [(number of G+C residues) x 4 °C]
Initial use Tm–5°C
Avoid internal hairpin structures
No secondary structure
Avoid a T at the 3’ end
Avoid overlapping 3’ ends – will form primer dimers
Can modify 5’ ends to add restriction sites
PRIMER DESIGN
Use specific
programs
OLIGO
Medprobe
PRIMER
DESIGNER
Sci. Ed software
Also available on the internet
http://www.hgmp.mrc.ac.uk/GenomeWeb/nuc-primer.html
Mg2+ CONCENTRATION
1
1.5
2
2.5
3
3.5
4 mM
Normally, 1.5mM MgCl2 is optimal
Best supplied as separate tube
Always vortex thawed MgCl2
Mg2+ concentration will be affected by the amount of DNA, primers and
nucleotides
USE MASTERMIXES WHERE POSSIBLE
How Powerful is PCR?
PCR can amplify a usable amount of DNA (visible by
gel electrophoresis) in ~2 hours.
 The template DNA need not be highly purified — a
boiled bacterial colony.
 The PCR product can be digested with restriction
enzymes, sequenced or cloned.
 PCR can amplify a single DNA molecule, e.g. from a
single sperm.

Applications of PCR





Amplify specific DNA sequences (genomic DNA, cDNA, recombinant
DNA, etc.) for analysis
1. Gene isolation
2. Fingerprint development
Introduce sequence changes at the ends of fragments
Rapidly detect differences in DNA sequences (e.g., length) for
identifying diseases or individuals
Identify and isolate genes using degenerate oligonucleotide primers
• Design mixture of primers to bind DNA encoding conserved protein
motifs
Genetic diagnosis - Mutation detection
The basis for many techniques to detect gene mutations (sequencing) 1/6 X 10-9 bp
Applications of PCR
Paternity testing
Mutagenesis to investigate protein function
Quantify differences in gene expression →
Reverse transcription (RT)-PCR
Identify changes in expression of unknown genes
→ Differential display (DD)-PCR
Forensic analysis at scene of crime
Industrial quality control
DNA sequencing
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

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: betagalactosidase screening, polylinker)
 Uses 32P-deoxynucleoside triphosphates

Sanger Method
in-vitro DNA synthesis using ‘terminators’, use of dideoxinucleotides 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
 Incorporation of di-deoxynucleotides into growing strand
terminates synthesis
 Synthesized strand sizes are determined for each dideoxynucleotide by using gel or capillary electrophoresis
 Enzymatic methods

Dideoxynucleotide
PPP
O
5’
CH2
O
BASE
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’
primer 5’
CCGTAC 5’
3’
dNTP
ddATP ddTTP
GGCA
ddCTP ddGTP
GGCAT
A
T C
GGC
G
G
GG
GGCATG
Chain Terminator Basics
Target
Template-Primer
TGCA
ddA
Extend
ddA
AddC
AC ddG
ACG ddT
ddT
ddC
ddG
Labeled Terminators
dN : ddN
100 : 1
Electrophoresis
Sanger Method Sequencing Gel
Sequencing of DNA by the Sanger method
Comparison

Sanger Method
• Enzymatic
• Requires DNA synthesis
• Termination of chain
elongation

Maxam Gilbert Method
• Chemical
• Requires DNA
• Requires long stretches of
DNA
• Breaks DNA at different
nucleotides