Biochemical Tool

ElectrophoresisHybridization

1. Molecules are separated by electric force2. F = qE : where q is net charge, E is electric field strength3. The velocity is encountered by friction4. qE = fv : where f is frictional force, v is velocity5. Therefore, mobility per unit field (U) = v/q = q/f = q/6pr :

where is viscosity of supporting medium, r is radius of sphere molecule

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E

F

f

v

q

Electrophoresis

Electro = flow of electricity,Phoresis= to carry across (from the Greek)

Definition

The separation of charged molecules using their different

rates of migration in an electrical field

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Samples

Separating Gel

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FACTORS INFLUENCING SEPARATION•Charge Density on Molecules -

Difference between pH •Molecular Size and Shape

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

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Electrophoresis

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

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

Electrophoresis

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

SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

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 ElectrophoresisApplication

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 sequenceCan 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 BlotWestern 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

Paper towel

Transfer membrane

Wet filter paperPaper towelweight

Western BlotWestern Blot

Add monoclonal antibodies

Rinse again

Antibodies will bind to specified protein

Stain the bound antibody for colour development

It should look like the gel you started with if a positive reaction occurred

• Block membrane e.g. dried nonfat milk

Rinse with ddH2O

Add antibody against yours with a marker (becomes the antigen)

Polymerase Chain Reaction (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

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 templatePrimers (anneal to flanking sequences)DNA polymerasedNTPsMg2+

Buffer

Can be performed by hand or in a machine called a thermal cycler.

1993: Nobel Prize for Chemistry

Background on PCR

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 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

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

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.

Example thermal cycler protocol used in lab:Step 1 7 min at 94˚C Initial DenatureStep 2 45 cycles of:

20 sec at 94˚C Denature 20 sec at 64˚C Anneal 1 min at 72˚C Extension

Step 3 7 min at 72˚C Final ExtensionStep 4 Infinite hold at 4˚C 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)

Starting nucleic acid - DNA/RNATissue, cells, blood, hair root, saliva, semen

Thermo-stable DNA polymerasee.g. Taq polymerase

OligonucleotidesDesign them well!

Buffer Tris-HCl (pH 7.6-8.0)

Mg2+

dNTPs (dATP, dCTP, dGTP, dTTP)

OPTIMISING PCR – THE REACTION COMPONENTS

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

RAW MATERIAL

Number of options available

Taq polymerasePfu polymeraseTth 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

POLYMERASE

Length ~ 18-30 nucleotides (21 nucleotides)

Base composition; 50 - 60% GC richpairs 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 structuresno 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

PRIMER DESIGN

Use specific programs

OLIGOMedprobe

PRIMERDESIGNERSci. Ed software

Also available on the internethttp://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 detectionbasis for many techniques to detect gene mutations (sequencing) - 1/6 X 10-9 bp

Paternity testing

Mutagenesis to investigate protein function

Quantify differences in gene expressionReverse transcription (RT)-PCR

Identify changes in expression of unknown genesDifferential display (DD)-PCR 

Forensic analysis at scene of crime

Industrial quality control

Applications of PCR

Sequencing of DNA by the Sanger method