Download Electrophoresis

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
-RER, cell membrane
-SER
-Cytosol
Cellular level
Microscope
Molecular level: Macromolecules
Proteins
Carbohydrates
Lipids
Nucleic acids
Atomic level
C, H, O, N, S, P
CONTENTS
Electrophoresis
Blotting and Hybridization
Polymerase Chain Reaction
DNA Sequences
Negatively-charged phosphate-sugar backbone
Various lengths
-
-
-
Specificity of nucleotides
Hydrogen bonds
DNA GEL ELECTROPHORESIS
1. For separating DNA strands of any size/length
2. Uses a gel to separate DNA strands
3. Uses electricity
Electrophoresis
 Molecules are separated by electric force
 F = qE : where q is net charge, E is electric field strength
 The velocity is encountered by friction
 qE = fv : where f is frictional force, v is velocity
 Therefore, mobility per unit field (U) = v/q = q/f = q/6pr : where 
is viscosity of supporting medium, r is radius of sphere molecule
E
v
F
-
f
q+
+---
+
Electrophoresis
Factors affected the mobility of molecules
-
1. Molecular factors
• Charge
• Size
• Shape
2. Environment factors
• Electric field strength
• Supporting media (pore: sieving effect)
• Running buffer
+
Electrophoresis
Electrophoresis
Types of supporting media
 Paper
 Agarose gel (Agarose gel electrophoresis)
 Polyacrylamide gel (PAGE)
 pH gradient (Isoelectric focusing electrophoresis)
 Cellulose acetate
Agarose Gel
Agarose:
 purified large MW
polysaccharide (from agar)
 very open (large pore) gel
 used frequently for large
DNA molecules
Agarose gel staining
Ethidium bromide
Electrophoresis
Polyacrylamide Gels
 Acrylamide polymer; very stable gel
 can be made at a wide variety of concentrations
 gradient 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 gm SDS/gm 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
Hybridization and
Blotting
Hybridization
Pairing of complementary DNA and/or RNA
Hybridization

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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
Oligonucleotide probes



Single stranded DNA (usually 15-40 bp)
Degenerate oligonucleotide 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



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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)
RNA Blotting (Northerns)



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)
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

Block membrane e.g. dried nonfat milk
Rinse with ddH2O
Add monoclonal
antibodies
Rinse again
Antibodies will bind to specified protein
Add antibody against yours with a marker (becomes the antigen)
Stain the bound antibody for colour development
It should look like the gel you
started with if a positive reaction
occurred
Polymerase Chain Reaction
(PCR)
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
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
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
Tissue, cells, blood, hair root, semen
 Thermo-stable DNA polymerase
e.g. Taq polymerase
 Oligonucleotides
Design them well!
 Buffer
Tris-HCl (pH 7.6-8.0)
Mg2+
dNTPs (dATP, dCTP, dGTP, dTTP)
RAW MATERIAL
 Organims, Organ, Tissue, cells ( blood, hair root, semen, 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
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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
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
 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