molecular techniques
DESCRIPTION
Molecular Techniques. Studies of cell Fractionation Purification/ Identification Structure/ Function. Proteins. Carbohydrates. Lipids. Nucleic acids. Organelle level. Cell fractionation Nucleus Mitochondria RER, cell membrane SER Cytosol. Cellular level. Microscope. - PowerPoint PPT PresentationTRANSCRIPT
Cellular level
Organelle level
Molecular level: Macromolecules
Atomic level
C, H, O, N, S, P
Microscope
Cell fractionation-Nucleus-Mitochondria-RER, cell membrane-SER-Cytosol
Proteins Carbohydrates Lipids Nucleic acids
Studies of cell-Fractionation-Purification/ Identification-Structure/ Function
CONTENTSCell fractionationElectrophoresis
Blotting and HybridizationPolymerase Chain Reaction
DNA Sequences
A lab technique which uses a centrifuge to separate the contents of a cell (organelles) into fractions, after the cell has
been gently lysed.
The process to break the cells is “HOMOGENIZATION” and the subsequent isolation of organelles is
“FRACTIONATION”.
The centrifugation technique is employed to isolate organelles regarding to their physical characteristics, e.g., size, shape and density. The methods frequently used are
“DIFFERENTIAL CENTRIFUGATION” and “DENSITY GRADIENT CENTRIFUGRATION”.
Cell fractionation
HOMOGENIZATIONCell lysis
Gently disrupt the cells to release cellular components Physical or non-physical cell lysis methods
Physical methods of cell disruption Disruption of cells results from the shearing forces generated between the cells and
either solid abrasive or liquid medium. Pastel and mortar homogenizer, Abrasive beads, Blender, Pressure
homogenization, Osmotic shock, Freezing/ thawing technique, Ultrasonification
Non-physical methods of cell disruption Organic solvents- to destroy membrane
Chaotropic anions- to destabilize membrane Detergents- to dissolve proteins and lipids membrane
Enzymatic digestion- to digest proteins, carbohydrates and lipids of cell wall
HOMOGENIZATION
FRACTIONATION
Centrifugation
Physical methods of cell disruption
1. Pastel and mortar homogenizer
2. Abrasive beads- sands, silica, alumina
3. Blender- special designed blades and chamber
4. Pressure homogenization- cells are imbibed with an inert gas (argon) which will form gas bubbles inside cytoplasm when the cells are suddenly returned to atmospheric pressure, hence rupture the membrane.
5. Osmotic shock- swelling and disrupting of cells in hypotonic solution
6. Freezing/ thawing technique- ice crystals rupture the cells
7. Ultrasonification- ultrasonic wave to break open the plasma membrane and leave the internal organelles intact.
Cell Fractionation
Non-physical methods of cell disruption
1. Organic solvents- chloroform/methanol mixtures can dissolve membrane lipids (destroy membranes) and release subcellular components.
2. Chaotropic anions- potassium thiocyanate, potassium bromide, lithium diiodosalicylate act to destabilize lipid membranes consequently, the subcellular components are being released.
3. Detergents- solubilize the integral membrane proteins by interacting with the phospholipid bilayer, e.g., SDS (anionic), Deoxycholate (non-denaturing) and Triton X-100 (non-ionic)
4. Enzymatic digestion- to digest proteins, carbohydrates and lipids of cell wall, Mixture of enzymes: chitinases, pectinases, lipases, proteases, cellulases
Cell Fractionation
Homogenization medium
Slightly hypo-osmotic or iso-osmotic – to preserve structural integrity of organelles
Osmoticums: sucrose, manitol, sorbitol
Chelating agents: EDTA or EGTA (remove Ca2+ or Mg2+ which are required by membrane proteases)
Protease inhibitor: endopeptidases, exopeptidases
The homogenization should be performed at 4oC to minimize protease activity
Cell Fractionation
Cell Fractionation
Fractionation The most widely used technique for fractionating cellular
components is centrifugation technique Particles of different density, size, and shape sediment at different
rate in a centrifugal field.
Factors affected the rate of sedimentation: particle size and shape the viscosity of suspending medium centrifugal field
* The particle remain stationary when the density of the particle and the density of the centrifugation medium are equal
Types of Centrifugation
1. Differential centrifugation
2. Rate-zonal centrifugation
3. Isopycnic centrifugation
Cell Fractionation
A centrifuge working at speeds in excess of 20,000 RPM is an
“ultracentrifuge”.
Differential centrifugation Separates particles as a function of size and
density
A particular centrifugal field is chosen over a period of time
Larger mass; lower centrifugation force; lesser spin time
Subjected to repeated steps with increasing of centrifugation force
Cell Fractionation
Differential centrifugation
Centrifugation force to pellet the cellular components
Cell componentsCell components Centrifugation force (x g)Centrifugation force (x g)NucleusNucleus 800-1,000800-1,000
Mitochondria, LysosomeMitochondria, LysosomeChloroplast, PeroxisomeChloroplast, Peroxisome
20,000-30,00020,000-30,000
RER membraneRER membrane 50,000-80,00050,000-80,000
Cell membrane,Cell membrane,SER membraneSER membrane
80,000-100,00080,000-100,000
RibosomeRibosome 150,000-300,000150,000-300,000
Cytosol fractionCytosol fraction SupernatantSupernatant
DifferentialCentrifugation
Pellet 1 Pellet 2 Pellet 4Pellet 3
Molecules separate according to size and shape
centrifugationRate-zonal centrifugation Medium
Slightly viscous Density gradient; a positive increment in density e.g., sucrose, GuHCl
Sample is applied on the top of density gradient
Particles separate into a series of bands (zone) in accordance to rate of sedimentation (S), size and shape
Rate-zonal Centrifugation
Rate-zonal Centrifugation
Centrifugation Fraction collection
Isopycniccentrifugation Based solely on the density of the particles
Separation medium– self-generating density gradient medium (CsCl medium)
Unaffected by the size or the shape of the particles
Mostly used to separate nucleic acids, large glycoproteins
Isopycnic Centrifugation
What make rate-zonal and isopycnic centrifugations difference?
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/6pr : where is viscosity of supporting medium, r is radius of sphere molecule
+ -+ - - -- +
E
F
f
v
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
Electrophoresis
Agarose Gel
purified large MW polysaccharide (from agar)
very open (large pore) gel
used frequently for large DNA molecules
Agarose gel stainingEthidium bromide
Fluorescence dye
Pounseur-S dye
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
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 charge)
- Second dimension is SDS-PAGE (separated by size)
- So called 2D-PAGE
- High throughput electrophoresis, high resolution
Electrophoresis
2-dimensional Gel Electrophoresis
Spot coordination pH MW
Hybridization and BlottingHybridization and Blotting
HybridizationHybridization
HybridizationHybridization Can be DNA:DNA, DNA:RNA, or RNA:RNA (RNA is easily Can be DNA:DNA, DNA:RNA, or RNA:RNA (RNA is easily
degraded)degraded) Dependent on the extent of complementationDependent on the extent of complementation Dependent on temperature, salt concentration, and solventsDependent on temperature, salt concentration, and solvents Small changes in the above factors can be used to discriminate Small changes in the above factors can be used to discriminate
between different sequences (e.g., small mutations can be detected)between different sequences (e.g., small mutations can be detected) Probes can be labeled with radioactivity, fluorescent dyes, enzymes, Probes can be labeled with radioactivity, fluorescent dyes, enzymes,
etc. etc. Probes can be isolated or synthesized sequencesProbes can be isolated or synthesized sequences
Oligonucleotide probesOligonucleotide probes Single stranded DNA (usually 15-40 bp)Single stranded DNA (usually 15-40 bp) Degenerate oligonucleotide probes can be used to Degenerate oligonucleotide probes can be used to
identify genes encoding characterized proteinsidentify genes encoding characterized proteins• Use amino acid sequence to predict possible DNA Use amino acid sequence to predict possible DNA
sequencessequences• Hybridize with a combination of probesHybridize with a combination of probes• TT(T/C) - TGG - ATG - GA(T/C) - TG(T/C) - could TT(T/C) - TGG - ATG - GA(T/C) - TG(T/C) - could
be used for FWMDC amino acid sequencebe used for FWMDC amino acid sequence Can specifically detect single nucleotide changesCan specifically detect single nucleotide changes
Detection of ProbesDetection of Probes Probes can be labeled with radioactivity, fluorescent dyes, Probes can be labeled with radioactivity, fluorescent dyes,
enzymes.enzymes. Radioactivity is often detected by X-ray film Radioactivity is often detected by X-ray film
(autoradiography)(autoradiography) Fluorescent dyes can be detected by fluorometers, Fluorescent dyes can be detected by fluorometers,
scannersscanners Enzymatic activities are often detected by the production of Enzymatic activities are often detected by the production of
dyes or light (x-ray film)dyes or light (x-ray film)
RNA Blotting (Northerns)RNA Blotting (Northerns) RNA is separated by size on a denaturing agarose gel and RNA is separated by size on a denaturing agarose gel and
then transferred onto a membrane (blot)then transferred onto a membrane (blot) Probe is hybridized to complementary sequences on the Probe is hybridized to complementary sequences on the
blot and excess probe is washed awayblot and excess probe is washed away Location of probe is determined by detection method (e.g., Location of probe is determined by detection method (e.g.,
film, fluorometerfilm, fluorometer))
Applications of RNA BlotsApplications of RNA BlotsDetect the expression level and transcript size of a Detect the expression level and transcript size of a
specific gene in a specific tissue or at a specific specific gene in a specific tissue or at a specific time. Sometimes mutations do not affect coding time. Sometimes mutations do not affect coding regions but transcriptional regulatory sequences regions but transcriptional regulatory sequences
(e.g., UAS/URS, promoter, splice sites, copy (e.g., UAS/URS, promoter, splice sites, copy number, transcript stability, etc.)number, transcript stability, etc.)
Western BlotWestern Blot Highly specific qualitative testHighly specific qualitative test Can determine if above or below thresholdCan determine if above or below threshold Typically used for researchTypically used for research Use denaturing SDS-PAGEUse denaturing SDS-PAGE
• Solubilizes, removes aggregates & adventitious proteins are Solubilizes, removes aggregates & adventitious proteins are eliminatedeliminated
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 milkBlock membrane e.g. dried nonfat milkRinse with ddH2O
Add antibody against yours with a marker (becomes the antigen)
Polymerase Chain ReactionPolymerase 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 manipulationAmplification 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
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.
Background on PCR
Three StepsThree Steps SeparationSeparation
Double Stranded DNA is denatured by heat into single strands. Double Stranded DNA is denatured by heat into single strands. Short Primers for DNA replication are added to the mixture.Short Primers for DNA replication are added to the mixture. PrimingPriming
DNA polymerase catalyzes the production of complementary new DNA polymerase catalyzes the production of complementary new strands.strands.
CopyingCopyingThe process is repeated for each new strand createdThe process is repeated for each new strand created
All three steps are carried out in the same vial but at different All three steps are carried out in the same vial but at different temperaturestemperatures
Step 1: SeparationStep 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 CofactorMagnesium as a CofactorStabilizes the reaction between:Stabilizes the reaction between:
• oligonucleotides and template DNAoligonucleotides and template DNA• DNA Polymerase and template DNADNA Polymerase and template DNA
Heat Denatures DNA by uncoiling the Double Helix strands.
Step 2: PrimingStep 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 PrimerSelecting a Primer Primer length Primer length Melting Temperature (Melting Temperature (TTmm) ) Specificity Specificity Complementary Primer Sequences Complementary Primer Sequences G/C content and Polypyrimidine (T, C) or G/C content and Polypyrimidine (T, C) or
polypurine (A, G) stretches polypurine (A, G) stretches 3’-end Sequence 3’-end Sequence Single-stranded DNASingle-stranded DNA
Step 3: PolymerizationStep 3: Polymerization Since the Taq polymerase works best at Since the Taq polymerase works best at
around 75 ° C (the temperature of the hot around 75 ° C (the temperature of the hot springs where the bacterium was springs where the bacterium was discovered), the temperature of the vial is discovered), the temperature of the vial is raised to 72-75 °Craised to 72-75 °C
The DNA polymerase recognizes the The DNA polymerase recognizes the primer and makes a complementary copy primer and makes a complementary copy of the template which is now single of the template which is now single stranded.stranded.
Approximately 150 nucleotides/secApproximately 150 nucleotides/sec
Potential Problems with TaqPotential Problems with Taq Lack of proof-reading of newly synthesized DNA.Lack of proof-reading of newly synthesized DNA.
Potentially can include di-Nucleotriphosphates (dNTPs) that Potentially can include di-Nucleotriphosphates (dNTPs) that are not complementary to the original strand. are not complementary to the original strand.
Errors in coding resultErrors in coding result Recently discovered thermostable DNA polymerases, Recently discovered thermostable DNA polymerases, Tth Tth
and and PfuPfu, are less efficient, yet highly accurate., are less efficient, yet highly accurate.
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.
How PCR works
Step 1 7 min at 94˚C Initial DenatureStep 2 45 cycles of:
20 sec at 94˚C Denature20 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
Thermal cycler protocol Example
The Polymerase Chain Reaction
PCR amplificationPCR amplification
Each cycle the oligo-nucleotide primers bind most all Each cycle the oligo-nucleotide primers bind most all templates due to the high primer concentrationtemplates due to the high primer concentration
The generation of mg quantities of DNA can be The generation of mg quantities of DNA can be achieved in ~30 cycles (~ 4 hrs)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 PCRTHE REACTION COMPONENTS
Organims, Organ, 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 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 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?How Powerful is PCR? PCR can amplify a usable amount of DNA (visible PCR can amplify a usable amount of DNA (visible
by gel electrophoresis) in ~2 hours.by gel electrophoresis) in ~2 hours. The template DNA need not be highly purified — a The template DNA need not be highly purified — a
boiled bacterial colony.boiled bacterial colony. The PCR product can be digested with restriction The PCR product can be digested with restriction
enzymes, sequenced or cloned.enzymes, sequenced or cloned. PCR can amplify a single DNA molecule, PCR can amplify a single DNA molecule, e.g.e.g. from from
a single sperm.a single sperm.
Applications of PCRApplications of PCR Amplify specific DNA sequences (genomic DNA, cDNA, recombinant DNA, Amplify specific DNA sequences (genomic DNA, cDNA, recombinant DNA,
etc.) for analysisetc.) for analysis1. Gene isolation1. Gene isolation2. Fingerprint development2. Fingerprint development
Introduce sequence changes at the ends of fragmentsIntroduce sequence changes at the ends of fragments Rapidly detect differences in DNA sequences (e.g., length) for identifying Rapidly detect differences in DNA sequences (e.g., length) for identifying
diseases or individualsdiseases or individuals Identify and isolate genes using degenerate oligonucleotide primersIdentify and isolate genes using degenerate oligonucleotide primers
• Design mixture of primers to bind DNA encoding conserved protein motifsDesign mixture of primers to bind DNA encoding conserved protein motifs
Genetic diagnosis - Mutation detectionGenetic diagnosis - Mutation detectionbasis for many techniques to detect gene mutations (sequencing) - 1/6 X 10basis for many techniques to detect gene mutations (sequencing) - 1/6 X 10 -9-9 bpbp
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
DNA sequencing
Applications of PCR
Sequencing of DNA by the Sanger method