Molecular Techniques
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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.

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Molecular Techniques

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Molecular techniques

Molecular Techniques


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

Molecular level: Macromolecules

Atomic level

C, H, O, N, S, P


Molecular techniques

CONTENTS

  • Cell fractionation

  • Electrophoresis

    • Blotting and Hybridization

    • Polymerase Chain Reaction

    • DNA Sequences


Molecular techniques

Cell fractionation

  • 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”.


Molecular techniques

HOMOGENIZATION

Cell lysis


Molecular techniques

HOMOGENIZATION

  • 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


Molecular techniques

FRACTIONATION

Centrifugation


Molecular techniques

Cell Fractionation

  • Physical methods of cell disruption

  • Pastel and mortar homogenizer

  • Abrasive beads-sands, silica, alumina

  • Blender-special designed blades and chamber

  • 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.

  • Osmotic shock-swelling and disrupting of cells in hypotonic solution

  • Freezing/ thawing technique-ice crystals rupture the cells

  • Ultrasonification- ultrasonic wave to break open the plasma membrane and leave the internal organelles intact.


Molecular techniques

Cell Fractionation

  • Non-physical methods of cell disruption

  • Organic solvents- chloroform/methanol mixtures can dissolve membrane lipids (destroy membranes) and release subcellular components.

  • Chaotropic anions- potassium thiocyanate, potassium bromide, lithium diiodosalicylate act to destabilize lipid membranes consequently, the subcellular components are being released.

  • 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)

  • Enzymatic digestion- to digest proteins, carbohydrates and lipids of cell wall, Mixture of enzymes: chitinases, pectinases, lipases, proteases, cellulases


Molecular techniques

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


Molecular techniques

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


Molecular techniques

Cell Fractionation

  • Types of Centrifugation

  • Differential centrifugation

  • Rate-zonal centrifugation

  • Isopycnic centrifugation

A centrifuge working at speeds in excess of 20,000 RPM is an “ultracentrifuge”.


Molecular techniques

Cell Fractionation

  • 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


Molecular techniques

Differential centrifugation

Centrifugation force to pellet the cellular components


Molecular techniques

Differential

Centrifugation

Pellet 1

Pellet 2

Pellet 3

Pellet 4


Molecular techniques

Molecules separate according to size and shape

centrifugation

Rate-zonal Centrifugation

  • Rate-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


Molecular techniques

Centrifugation

Fraction collection

Rate-zonal Centrifugation


Molecular techniques

Isopycnic Centrifugation

  • Isopycnic

  • centrifugation

  • 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


Molecular techniques

What make rate-zonal and isopycnic centrifugations difference?


Molecular techniques

Electrophoresis

E

v

F

+ -

+ - - -

-

+

q

f

  • 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


Molecular techniques

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

+


Molecular techniques

Electrophoresis


Molecular techniques

Electrophoresis

  • Types of supporting media

  • Paper

  • Agarose gel (Agarose gel electrophoresis)

  • Polyacrylamide gel (PAGE)

  • pH gradient (Isoelectric focusing electrophoresis)

  • Cellulose acetate


Molecular techniques

  • Agarose Gel

  • purified large MW polysaccharide (from agar)

  • very open (large pore) gel

  • used frequently for large DNA molecules


Molecular techniques

Electrophoresis

Agarose gel staining

Ethidium bromide

Fluorescence dye

Pounseur-S dye


Molecular techniques

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)


Molecular techniques

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


Molecular techniques

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


Molecular techniques

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


Molecular techniques

2-dimensional Gel Electrophoresis

  • Spot coordination

  • pH

  • MW


Hybridization and blotting

Hybridization and Blotting


Hybridization

Hybridization


Hybridization1

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

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

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 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

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

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

Paper towel

Wet filter paper

Transfer membrane

Paper towel


Western blot1

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

Western Blot

  • Block membrane e.g. dried nonfat milk

Rinse with ddH2O

Add monoclonal antibodies

Rinse again

Antibodies will bind to specified protein


Polymerase chain reaction pcr

Polymerase Chain Reaction(PCR)


Molecular techniques

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


Molecular techniques

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

  • 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.


Three steps

Three Steps

  • Separation

    Double Stranded DNA is denatured by heat into single strands.

  • Short Primers for DNA replication are added to the mixture.

  • Priming

    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

Step 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 Cofactor

Stabilizes the reaction between:

  • oligonucleotides and template DNA

  • DNA Polymerase and template DNA


Molecular techniques

Heat

Denatures DNA by uncoiling the Double Helix strands.


Step 2 priming

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

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

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

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.


Molecular techniques

How PCR works

Begins with DNA containing a sequence to be amplified and a pair of synthetic oligonucleotide primers that flank the sequence.

Next, denature the DNA at 94˚C.

Rapidly cool the DNA (37-65˚C) and anneal primers to complementary s.s. sequences flanking the target DNA.

Extend primers at 70-75˚C using a heat-resistant DNA polymerase (e.g., Taq polymerase derived from Thermus aquaticus).

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.

Extend the primers at 70-75˚C once more to allow incomplete extension products in the reaction mixture to extend completely.

Cool to 4˚C and store or use amplified PCR product for analysis.


Molecular techniques

Thermal cycler protocol Example

Step 1 7 min at 94˚CInitial Denature

Step 2 45 cycles of:

20 sec at 94˚CDenature

20 sec at 64˚CAnneal

1 min at 72˚CExtension

Step 3 7 min at 72˚CFinal Extension

Step 4 Infinite hold at 4˚CStorage


Molecular techniques

The Polymerase Chain Reaction


Pcr amplification

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)


Molecular techniques

OPTIMISING PCR

THE REACTION COMPONENTS

  • 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)


Molecular techniques

RAW MATERIAL

  • 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


Molecular techniques

POLYMERASE

  • 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


Molecular techniques

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 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


Molecular techniques

PRIMER DESIGN

Use specific programs

OLIGOMedprobe

PRIMERDESIGNERSci. Ed software

Also available on the internet

http://www.hgmp.mrc.ac.uk/GenomeWeb/nuc-primer.html


Molecular techniques

1 1.5 2 2.5 3 3.5 4 mM

Mg2+ CONCENTRATION

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


Molecular techniques

USE MASTERMIXES WHERE POSSIBLE


How powerful is pcr

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

Applications of PCR

  • Amplify specific DNA sequences (genomic DNA, cDNA, recombinant DNA, etc.) for analysis

    1. Gene isolation

    2. Fingerprint development

  • 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


Molecular techniques

Applications of PCR

  • 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


Molecular techniques

Sequencing of DNA by the Sanger method


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