Biochemical Tool
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Transcript of Biochemical Tool
Biochemical ToolElectrophoresisHybridization
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 DNA3. 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 probesSingle 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 ProbesProbes can be labeled with radioactivity,
fluorescent dyes, enzymes.Radioactivity is often detected by X-ray
film (autoradiography)Fluorescent dyes can be detected by
fluorometers, scannersEnzymatic 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 milkRinse 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: SeparationCombine Target Sequence, DNA primers
template, dNTPs, Taq PolymeraseTarget 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, semenObtain the best starting material you can.Some can contain inhibitors of PCR, so they must be
removed e.g. Haem in bloodGood quality genomic DNA if possibleBlood – consider commercially available reagents
Qiagen– expense?Empirically determine the amount to add
RAW MATERIAL
Number of options availableTaq 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 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°CAvoid internal hairpin structures
no secondary structureAvoid a T at the 3’ endAvoid overlapping 3’ ends – will form primer dimersCan 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 MgCl2Mg2+ 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
Paternity testingMutagenesis to investigate protein functionQuantify differences in gene expression
Reverse transcription (RT)-PCRIdentify changes in expression of unknown genes
Differential display (DD)-PCR Forensic analysis at scene of crimeIndustrial quality control
Applications of PCR
Sequencing of DNA by the Sanger method