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

HOMOGENIZATION

Cell 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 staining

Ethidium 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 between different sequences (e.g., small mutations can be detected)detected)

Probes can be labeled with radioactivity, fluorescent dyes, Probes can be labeled with radioactivity, fluorescent dyes, enzymes, etc. enzymes, 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 Blots

Detect 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 milk

Rinse 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

SeparationSeparationDouble 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 PCR

THE 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 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?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