Transcript Biochemical Tool Electrophoresis Hybridization Electrophoresis Electro = flow of electricity, Phoresis= to carry across (from the Greek) 1. 2. 3. 4. 5. Molecules are separated by electric force F = qE.

Biochemical Tool
Electrophoresis
Hybridization
Electrophoresis
Electro = flow of electricity,
Phoresis= to carry across (from the Greek)
1.
2.
3.
4.
5.
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
+q
+---
F
+
Definition
The separation of charged molecules
using their different rates of migration
in an electrical field
FACTORS INFLUENCING
SEPARATION
•Charge Density on Molecules Difference between pH
•Molecular Size and Shape
Samples
Separating Gel
+
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
+
Types of supporting media
 Paper
 Agarose gel (Agarose gel electrophoresis)
 Polyacrylamide gel (PAGE)
 pH gradient (Isoelectric focusing electrophoresis)
 Cellulose acetate
Gel electrophoresis
 A gel is a colloid, a suspension of tiny
particles in a medium, occurring in a solid
form, like gelatin
 Gel electrophoresis refers to the separation
of charged particles located in a gel when an
electric current is applied
 Charged particles can include DNA, amino
acids, peptides
Poliakrialimida
• Polimer dari akrilamid
• Pori-porinya lebih kecil dari polimer
agarosa
• Menghasilkan tingkat resolusi yang lebih
tinggi
• Gel dibuat dengan menggunakan 2
lembaran kaca atau plastik mika
Poliakrialimida
 Penyangga: TBE
 Kegunaan:
1. Memisahkan DNA berukuran kecil (AFLP, SNP)
2. mengurutkan DNA
3. Memisahkan protein (perlu ditambah SDS,
sehingga disebut SDS-PAGE: SDS poly
acrylamide Gel Electrophoresis)
Pembuatan gel poliakrialimida
• Akrilamida + metilen bis akrilamida
• Ukuran pori ditentukan dengan
menentukan konsentrasi akrilamida dan
metilen bis akrilamidanya
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)
Electrophoresis
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
Electrophoresis
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
Electrophoresis
2-dimensional Gel Electrophoresis
-
First dimension is IFE (separated by pI)
-
Second dimension is SDS-PAGE
(separated by size)
-
So called 2D-PAGE
-
High throughput electrophoresis, high
resolution
-
Core methods for “Proteomics”
2-dimensional Gel Electrophoresis
Spot coordination
- pI
- MW
2-dimensional Gel Electrophoresis
Application
Hybridization and
Blotting
Hybridization
Hybridization
 Can be DNA:DNA, DNA:RNA, or RNA:RNA (RNA is easily
degraded)
 Dependent on the extent of complementation
 Dependent 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, etc.
 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
 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)
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, etc.)
Western Blot
•
•
•
•
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
 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.
Background on PCR
PCR, “discovered” in 1983 by Kary Mullis
DNA template
Primers
(anneal to flanking sequences)
DNA polymerase
dNTPs
Mg2+
Buffer
Can be performed by hand or in a machine called a
thermal cycler.
1993: Nobel Prize for Chemistry
Three Steps
 Separation: Double Stranded DNA is denatured by heat
into single strands.
 Short Primers for DNA replication are added to the
mixture.
 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: Usually fewer than 3000 bp
– Identified by a specific pair of DNA primersusually 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
 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
°
 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 2045 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.
Example thermal cycler protocol
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
used in lab:
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,
saliva, 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
 Tissue, cells, blood, hair root, saliva, semen
 Obtain the best starting material you can.
 Some can contain inhibitors of PCR, so they must be
removed e.g. Haem in blood
 Good quality genomic DNA if possible
 Blood – consider commercially available reagents
Qiagen– expense?
 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’
2. Cloning
5' proofreading exonuclease activity
PRIMER DESIGN
Length ~ 18-30 nucleotides (21 nucleotides)
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
 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
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
Sequencing of DNA by
the Sanger method