Reminder: All molecular techniques are based

on the chemical “personality” (or chemical properties) of the DNA molecule (or nucleic

acids)

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

CONTENTSElectrophoresis

Blotting and HybridizationPolymerase Chain Reaction

DNA Sequences

Negatively-charged phosphate-sugar backbone

-- -

-

Hydrogen bonds

Specificity of nucleotides

Various lengths

DNA GEL ELECTROPHORESIS1.For separating DNA strands of any size/length2.Uses a gel to separate DNA strands3.Uses electricity

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:

purified large MW polysaccharide (from agar)

very open (large pore) gel

used frequently for large DNA molecules

Agarose Gel

Agarose gel staining

Ethidium bromide

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)

- Separate molecules according to their isoelectric point (pI)

- At isoelectric point (pI) molecule has no charge (q=0), hence molecule ceases

- pH gradient medium

Isoelectric Focusing Electrophoresis (IFE)

- First dimension is IFE (separated by charge)

- Second dimension is SDS-PAGE (separated by size)

- So called 2D-PAGE

- High throughput electrophoresis, high resolution

2-dimensional Gel Electrophoresis

2-dimensional Gel Electrophoresis

Spot coordination pH MW

Hybridization and BlottingHybridization and Blotting

HybridizationHybridization Pairing of complementary DNA and/or RNA

HybridizationHybridization It can be DNA:DNA, DNA:RNA, or RNA:RNA (RNA

is easily degraded) It depended on the extent of complementation It depended 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.

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 sequence

Can specifically detect single nucleotide changes

Detection of Probes

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

Radioactivity is often detected by X-ray Radioactivity is often detected by X-ray film (autoradiography)film (autoradiography)

Fluorescent dyes can be detected by Fluorescent dyes can be detected by fluorometers, scannersfluorometers, scanners

Enzymatic activities are often detected by Enzymatic activities are often detected by the production of dyes or light (x-ray film)the production of dyes or light (x-ray film)

RNA Blotting (Northerns)

RNA is separated by size on a denaturing RNA is separated by size on a denaturing agarose gel and then transferred onto a agarose gel and then transferred onto a membrane (blot)membrane (blot)

Probe is hybridized to complementary Probe is hybridized to complementary sequences on the blot and excess probe is sequences on the blot and excess probe is washed awaywashed away

Location of probe is determined by Location of probe is determined by detection method (e.g., film, fluorometerdetection method (e.g., film, fluorometer))

Western BlotWestern Blot Protein blottingProtein blotting 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 Solubilizes, removes aggregates & adventitious proteins are eliminatedproteins are eliminated

Components of the gel are then transferred to a solid support or transfer membrane

Paper towel

Transfer membrane

Wet filter paper

Paper 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

The 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

SeparationDouble Stranded DNA is denatured by heat into single strands. Short Primers for DNA replication are added to the mixture.

PrimingDNA polymerase catalyzes the production of complementary new strands.

CopyingThe process is repeated for each new strand createdAll three steps are carried out in the same vial but at different temperatures

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 Cofactor Mg stabilizes the reaction between:

•oligonucleotides and template DNA•DNA Polymerase and template DNA

Heat Denatures DNA by uncoiling the Double Helix strands.

Step 2: PrimingStep 2: Priming Decrease temperature by 15-25 °C

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 Since the Taq polymerase works best at around 75 ° C (the best at around 75 ° C (the temperature of the hot springs temperature of the hot springs where the bacterium was where the bacterium was discovered), the temperature of the discovered), the temperature of the vial is raised to 72-75 °Cvial is raised to 72-75 °C

The DNA polymerase recognizes The DNA polymerase recognizes the primer and makes a the primer and makes a complementary copy of the complementary copy of the template which is now single 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 Potentially can include di-Nucleotriphosphates

(dNTPs) that are not complementary to the (dNTPs) that are not complementary to the original strand. original strand.

Errors in coding resultErrors in coding result Recently discovered thermostable DNA Recently discovered thermostable DNA

polymerases, polymerases, Tth Tth and and PfuPfu, are less efficient, yet , are less efficient, yet highly accuratehighly 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 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,

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, semen, callus, leaves, root, seed)

Obtain the best starting material.

Some can contain inhibitors of PCR, so they must be removed e.g. Haem in blood

Good quality genomic DNA if possible

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

3. Marker development

POLYMERASE

Length ~ 10-30 nucleotides (21 nucleotides for gene isolation)

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 PCR can amplify a usable amount of DNA (visible by gel electrophoresis) in ~2 (visible by gel electrophoresis) in ~2

hours.hours. The template DNA need not be highly The template DNA need not be highly

purified — a boiled bacterial colony.purified — a boiled bacterial colony. The PCR product can be digested with The PCR product can be digested with restriction enzymes, sequenced or cloned.restriction enzymes, sequenced or cloned.

PCR can amplify a single DNA molecule, PCR can amplify a single DNA molecule, e.g.e.g. from a single sperm. from a single sperm.

Applications of PCRApplications of PCR Amplify specific DNA sequences (genomic DNA, cDNA, Amplify specific DNA sequences (genomic DNA, cDNA,

recombinant DNA, etc.) for analysisrecombinant DNA, etc.) for analysis

1. Gene isolation1. Gene isolation

2. 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., Rapidly detect differences in DNA sequences (e.g.,

length) for identifying diseases or individualslength) for identifying diseases or individuals Identify and isolate genes using degenerate Identify and isolate genes using degenerate

oligonucleotide primersoligonucleotide primers• Design mixture of primers to bind DNA encoding Design mixture of primers to bind DNA encoding

conserved protein motifsconserved protein motifs

Genetic diagnosis - Mutation detectionGenetic diagnosis - Mutation detectionThe basis for many techniques to detect gene The basis for many techniques to detect gene mutations (sequencing) - 1/6 X 10mutations (sequencing) - 1/6 X 10-9-9 bp bp

Paternity testing

Mutagenesis to investigate protein function

Quantify differences in gene expression →Reverse transcription (RT)-PCR

Identify changes in expression of unknown genes→ Differential display (DD)-PCR 

Forensic analysis at scene of crime

Industrial quality control

DNA sequencing

Applications of PCR

DNA SequencingDNA Sequencing

DNA sequencingDNA sequencing Determination of nucleotide sequenceDetermination of nucleotide sequence

the determination of the precise sequence of the determination of the precise sequence of nucleotides in a sample of DNAnucleotides in a sample of DNA

Two similar methods:Two similar methods:1. Maxam and Gilbert method1. Maxam and Gilbert method

2. Sanger method2. Sanger method

They depend on the production of a mixture of They depend on the production of a mixture of oligonucleotides labeled either radioactively or oligonucleotides labeled either radioactively or

fluorescein, with one common end and differing in fluorescein, with one common end and differing in length by a single nucleotide at the other endlength by a single nucleotide at the other end

This mixture of oligonucleotides is separated by This mixture of oligonucleotides is separated by high resolution electrophoresis on polyacrilamide high resolution electrophoresis on polyacrilamide

gels and the position of the bands determinedgels and the position of the bands determined

The Maxam-Gilbert The Maxam-Gilbert TechniqueTechnique

Principle: Principle: Chemical Degradation of Chemical Degradation of PurinesPurines• Purines (A, G) damaged by Purines (A, G) damaged by

dimethylsulfatedimethylsulfate• Methylation of baseMethylation of base• Heat releases baseHeat releases base• Alkali cleaves GAlkali cleaves G• Dilute acid cleave A>GDilute acid cleave A>G

Maxam-Gilbert Maxam-Gilbert TechniqueTechnique

•Pyrimidines (C, Pyrimidines (C, T) are damaged T) are damaged by hydrazineby hydrazine

•Piperidine Piperidine cleaves the cleaves the backbonebackbone

•2 M NaCl inhibits 2 M NaCl inhibits the reaction with the reaction with TT

Maxam and Gilbert MethodMaxam and Gilbert Method Chemical degradation of purified fragments (chemical degradation)Chemical degradation of purified fragments (chemical degradation) The single stranded DNA fragment to be sequenced is end-labeled The single stranded DNA fragment to be sequenced is end-labeled

by treatment with alkaline phosphatase to remove the 5’phosphateby treatment with alkaline phosphatase to remove the 5’phosphate It is then followed by reaction with P-labeled ATP in the presence of It is then followed by reaction with P-labeled ATP in the presence of

polynucleotide kinase, which attaches P labeled to the 5’terminalpolynucleotide kinase, which attaches P labeled to the 5’terminal The labeled DNA fragment is then divided into four aliquots, each of The labeled DNA fragment is then divided into four aliquots, each of

which is treated with a reagent which modifies a specific basewhich is treated with a reagent which modifies a specific base1. Aliquot A + dimethyl sulphate, which methylates guanine residue1. Aliquot A + dimethyl sulphate, which methylates guanine residue2. Aliquot B + formic acid, which modifies adenine and guanine residues2. Aliquot B + formic acid, which modifies adenine and guanine residues3. Aliquot C + Hydrazine, which modifies thymine + cytosine residues3. Aliquot C + Hydrazine, which modifies thymine + cytosine residues4. Aliquot D + Hydrazine + 5 mol/l NaCl, which makes the reaction specific for 4. Aliquot D + Hydrazine + 5 mol/l NaCl, which makes the reaction specific for cytosinecytosine The four are incubated with piperidine which cleaves the sugar The four are incubated with piperidine which cleaves the sugar

phosphate backbone of DNA next to the residue that has been phosphate backbone of DNA next to the residue that has been modifiedmodified

Maxam-Gilbert Maxam-Gilbert sequencing - modificationssequencing - modifications

Maxam-Gilbert sequencing: Summary

Advantages/disadvantagesMaxam-Gilbert sequencing

Requires lots of purified DNA, and many intermediate purification steps Relatively short readings

Automation not available (sequencers) Remaining use for ‘footprinting’ (partial protection

against DNA modification when proteins bind to specific regions, and that produce ‘holes’ in the

sequence ladder)

In contrast, the Sanger sequencing methodology requires little if any DNA

purification, no restriction digests, and no labeling of the DNA sequencing template

SangerSanger Fred Sanger, 1958Fred Sanger, 1958

• Was originally a Was originally a protein chemistprotein chemist

• Made his first mark Made his first mark in sequencing in sequencing proteinsproteins

• Made his second Made his second mark in sequencing mark in sequencing RNARNA

1980 dideoxy 1980 dideoxy sequencingsequencing

Original Sanger MethodOriginal Sanger Method Random incorporation of a dideoxynucleoside Random incorporation of a dideoxynucleoside

triphosphate into a growing strand of DNAtriphosphate into a growing strand of DNA Requires DNA polymerase IRequires DNA polymerase I

Requires a cloning vector with initial primer Requires a cloning vector with initial primer (M13, high yield bacteriophage, modified by (M13, high yield bacteriophage, modified by

adding: beta-galactosidase screening, adding: beta-galactosidase screening, polylinker)polylinker)

Uses Uses 3232P-deoxynucleoside triphosphatesP-deoxynucleoside triphosphates

Sanger MethodSanger Method

in-vitro DNA synthesis using ‘terminators’, use of in-vitro DNA synthesis using ‘terminators’, use of dideoxi- nucleotides that do not permit chain dideoxi- nucleotides that do not permit chain

elongation after their integration elongation after their integration DNA synthesis using deoxy- and DNA synthesis using deoxy- and

dideoxynucleotides that results in termination of dideoxynucleotides that results in termination of synthesis at specific nucleotidessynthesis at specific nucleotides

Requires a primer, DNA polymerase, a template, a Requires a primer, DNA polymerase, a template, a mixture of nucleotides, and detection systemmixture of nucleotides, and detection system

Incorporation of di-deoxynucleotides into growing Incorporation of di-deoxynucleotides into growing strand terminates synthesisstrand terminates synthesis

Synthesized strand sizes are determined for each Synthesized strand sizes are determined for each di-deoxynucleotide by using gel or capillary di-deoxynucleotide by using gel or capillary

electrophoresiselectrophoresis Enzymatic methodsEnzymatic methods

DideoxynucleotideDideoxynucleotide

no hydroxyl group at 3’ endprevents strand extension

CH2O

OPPP5’

3’

BASE

The principlesThe principles Partial copies of DNA fragments made

with DNA polymerase Collection of DNA fragments that

terminate with A,C,G or T using ddNTP Separate by gel electrophoresis

Read DNA sequence

CCGTAC3’ 5’5’ 3’primer

dNTP

ddATP

GGCA

ddTTP

GGCAT

ddCTP

GGC G

ddGTP

GGGGCATG

A T C G

Chain Terminator BasicsChain Terminator Basics

TargetTemplate-Primer

ExtendddA

ddG

ddC

ddTLabeled Terminators

ddA

AddC

ACddG

ACG ddT

TGCA

dN : ddN100 : 1

ElectrophoresisElectrophoresis

Sanger Method Sequencing GelSanger Method Sequencing Gel

Sequencing of DNA by the Sanger method

ComparisonComparison

Sanger MethodSanger Method• EnzymaticEnzymatic• Requires DNA Requires DNA

synthesissynthesis• Termination of Termination of

chain elongationchain elongation

Maxam Gilbert Maxam Gilbert MethodMethod• ChemicalChemical• Requires DNARequires DNA• Requires long Requires long

stretches of DNAstretches of DNA• Breaks DNA at Breaks DNA at

different nucleotidesdifferent nucleotides