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 levelC, H, O, N, S, P

Microscope

Cell fractionation-Nucleus-Mitochondria-Ceell membrane-Cytosol

Proteins Carbohydrates Lipids Nucleic acids

Studies of cell-Fractionation-Purification/ Identification-Structure/ Function

Negatively-charged phosphate-sugar backbone

-- -

-

Hydrogen bonds

Specificity of nucleotides

Various lengths

Nucleic Acids

CONTENTSEnzymes

ElectrophoresisBlotting and HybridizationPolymerase Chain Reaction

DNA Sequences

Enzymes

Large molecules made of various amino acids

Act as catalysts to speed up reactions w/out being destroyed• Increase the rate of reaction• Highly specific• Lowers energy of activation level• Activity lost if denatured• May contain cofactors such as metal ions or

organic (vitamins)

66

Name of Enzymes

End in –ase Identifies a reacting substance

1. sucrase – reacts sucrose 2. lipase - reacts lipid Describes function of enzyme

1. oxidase – catalyzes oxidation2. hydrolase – catalyzes hydrolysis

Classification of EnzymesClass Reactions catalyzed Oxidoreductoases oxidation-reduction Transferases transfer group of atoms Hydrolases hydrolysis Lyases add/remove atoms to/from a

double bond Isomerases rearrange atoms Ligases combine molecules

88

Enzyme Action: Lock and Key Model

An enzyme binds a substrate in a region called the active site

Only certain substrates can fit the active site Amino acid R groups in the active site help substrate

bind Enzyme-substrate complex forms Substrate reacts to form product Product is released

Lock and Key Model

++ + +

E + S ES complex E + P

S

P

P S

Enzymes use in Molecular Genetics

1. Restriction endonucleases/enzymes

2. Ligase 3. DNA polymerase

Restriction EnzymesRestriction EnzymesMolecular scissors which isolated from bacteria where

they are used as Bacterial defense against viruses

Molecular scalpels to cut DNA in a precise and predictable manner

Enzyme produced by bacteria that typically recognize specific 4-8 base pair sequences called restriction

sites, and then cleave both DNA strands at this site

A class of endo-nucleases that cleavage DNA after recognizing a specific sequence

Members of the class of nucleases

Breaking the phosphodiester bonds that link adjacent nucleotides in DNA and

RNA molecules

EndonucleaseCleave nucleic acids at internal position

ExonucleaseProgressively digest from the ends of the nucleic acid molecules

Nuclease

EndonucleaseType CharacteristicsI Have both restriction and modification activity

Cut at sites 1000 nucleotides or more away from recognition site

ATP is requiredII It has only restriction site activity

Its cut is predictable and consistent manner at a site within or adjacent to restriction site

It require only magnesium ion as cofactor III Have both restriction and modification activity

Cut at sites closed to recognition site ATP is required

There are already more than 1200 type II enzymes isolated from prokaryotic organism

They recognize more than 130 different nucleotide sequence

They scan a DNA molecule, stopping only when it recognizes a specific sequence of nucleotides that are composed of symetrical, palindromic sequence

Palindromic sequence:The sequence read forward on one DNA strand is identical to the sequence read in the opposite direction on the complementary strand

To Avoid confusion, restriction endo-nucleases are named according to the following nomenclature

Restriction Enzymes

The first letter is the initial letter of the genus name of the organism from which the enzyme is isolated

The second and third letters are usually the initial letters of the organisms species name. It is written in italic

A fourth letter, if any, indicates a particular strain organism

Originally, roman numerals were meant to indicate the order in which enzymes, isolated from the same organisms and strain, are eluted from a chromatography column. More often, the roman numerals indicate the order of discovery

Nomenclature

NomenclatureEcoEcoRIRI E : Genus EscherichiaE : Genus Escherichia

co: Species colico: Species coliR : Strain RY13R : Strain RY13I : First endonuclease isolatedI : First endonuclease isolated

BamBamHIHI B : Genus BacillusB : Genus Bacillusam: species amyloliquefaciensam: species amyloliquefaciensH : Strain HH : Strain HI : First endonuclease isolatedI : First endonuclease isolated

HinHindIIIdIII H : Genus HaemophilusH : Genus Haemophilusin : species influenzaein : species influenzaed : strain Rdd : strain RdIII : Third endonuclease isolatedIII : Third endonuclease isolated

SpecificityEnzymeEnzyme SourceSource SequenceSequence EndEndBamHIBamHI Bacillus Bacillus

amyloliquefaciens Hamyloliquefaciens HGGGATCCGATCC StickStick

yyBglIIBglII Bacillus globigiiBacillus globigii AAGATCTGATCT StickStick

yyEcoRIEcoRI Escherichia coli RY13Escherichia coli RY13 GGAATTCAATTC StickStick

yyEcoRIIEcoRII Escherichia coli R245Escherichia coli R245 CCTGGCCTGG StickStick

yyHaeIIIHaeIII Haemophilus aegyptiusHaemophilus aegyptius GGGGCCCC BluntBluntHindIIHindII Haemophilus influenzae Haemophilus influenzae

RdRdGTPyGTPyPuACPuAC BluntBlunt

HindIIIHindIII Haemophilus influenzae Haemophilus influenzae RdRd

AAAGCTTAGCTT StickStickyy

HpaIIHpaII Haemophilus Haemophilus parainfluenzaeparainfluenzae

CCCGGCGG StickStickyy

NotINotI Nocardia otitidis-Nocardia otitidis-caviarumcaviarum

GCGCGGCCGGGCCGCC

StickStickyy

PstIPstI Providencia stuartii 164Providencia stuartii 164 CTGCACTGCAGG StickStickyy

Restriction Product

Restriction enzymesRestriction enzymes

degenerate or specific sequences

kind of ends produced (5’ or 3’ overhang (cohesive=sticky), blunt=flush)

number of nucleotides recognized (4, 6,8 base-cutters most common)

whether cleavage occurs within the recognition sequence

Restriction enzymes can be grouped by:

A restriction enzyme (A restriction enzyme (EcoEcoRI)RI)1. 6-base cutter

4. produces a 5’ overhang (sticky end)

2. Specific palindromic sequence (5’GAATTC) 3. Cuts within the recognition sequence (type II enzyme)

Any of a class of enzymes that act as catalysts in

chemical reactions in which molecules are linked

together, as in the synthesis and repair of DNA or in the formation of recombinant

DNA

Any of a class of enzymes that catalyze the linkage of two molecules, generally

utilizing ATP as the energy donor (synthetase).

Ligase

Function of DNA ligaseThe enzyme, DNA ligase, repairs the millions of DNA breaks generated during the normal course of a cell's life, for example, linking together the abundant DNA fragments formed during replication of the genetic material in dividing cells.

EC 6 LigasesEC 6.1 Forming carbon—oxygen

bondsEC 6.2 Forming carbon—sulfur

bondsEC 6.3 Forming carbon—nitrogen

bondsEC 6.4 Forming carbon—carbon

bondsEC 6.5 Forming phosphoric ester

bondsEC 6.6 Forming nitrogen—metal

bonds

Ligase

DNA Ligase Mechanism

DNA Ligase Mechanism

Human DNA ligase III gene encodes both nuclear and mitochondrial enzymes.

DNA ligase plays a central role in DNA replication, recombination, and DNA repair.

Human DNA Ligase

DNA Polymerase an enzyme that is template

based and has both 5’->3' DNA polymerase activity and 3’->5' exonuclease activity.

highly processive, meaning it synthesizes long stretches of DNA without dissociating from the DNA template.

an open right hand, an open right hand, composed of a thumb composed of a thumb domain that binds to domain that binds to thioredoxin, a finger thioredoxin, a finger domain in which catalytic domain in which catalytic activity resides, a palm activity resides, a palm domain that cradles the domain that cradles the DNA, and a terminal DNA, and a terminal exonuclease domain exonuclease domain 

Three main features of the DNA synthesis reaction

1. DNA polymerase I catalyzes formation of phosphodiester bond between 3’-OH of the deoxyribose (on the last nucleotide) and he 5’-phosphate of the dNTP.Energy for this reaction is derived from the release of two of the three phosphates of the dNTP.

2. DNA polymerase “finds” the correct complementary dNTP at each step in the lengthening process.

• rate ≤ 800 dNTPs/second• low error rate

3. Direction of synthesis is 5’ to 3’

DNA elongation

DNA elongation

Types of DNA polymerasePolymerase Polymerization Exonuclease Exonuclease

#Copies (5’-3’) (3’-5’) (5’-3’)

I Yes Yes Yes400II Yes Yes No ?III Yes Yes No 10-20•3’ to 5’ exonuclease activity : ability to remove nucleotides from the

3’ end of the chain•Important proofreading ability •Without proofreading error rate (mutation rate) is 1 x 10-6

•With proofreading error rate is 1 x 10-9 (1000-fold decrease)

•5’ to 3’ exonuclease activity : the ability in DNA replication & repair.

Eukaryotic enzymesFive common DNA polymerases from mammals.

1. Polymerase (alpha): nuclear, DNA replication, no proofreading

2. Polymerase (beta): nuclear, DNA repair, no proofreading

3. Polymerase (gamma): mitochondria, DNA repl., proofreading

4. Polymerase (delta): nuclear, DNA replication, proofreading

5. Polymerase (epsilon): nuclear, DNA repair (?), proofreading

• Different polymerases for the nucleus and mtDNA

• Some polymerases proofread; others do not.• Some polymerases used for replication; others

for repair.• Polymerases vary by species.

In this illustration, DNA ligase (in color) encircles the DNA double helix.

Researchers investigating an important DNA-repair enzyme now have a better picture of the final steps of a process that glues together, or ligates, the ends of DNA strands to restore the double helix.

The enzyme, DNA ligase, repairs the millions of DNA breaks generated during the normal course of a cell's life, for example, linking together the abundant DNA fragments formed during replication of the genetic material in dividing cells.

GEL ELECTROPHORESIS

1. For separating disperse charged biological molecule of any size/length

2. Uses electricity3. Uses a matrix 4. Uses buffer solution

First observed by Reuss, 1807

The motion of disperse charged particle relatives to a fluid under the influence of a spatially uniform electric field

Factors affecting the mobility of molecules:

1. Molecular factors• Charge• Size• Shape

2. Environment factors• Electric field strength• Matrix (pore: sieving effect)• Running buffer

-

+

Electrophoresis

Electrophoresis

Paper Agarose

1. purified large MW polysaccharide (from agar)2. very open (large pore) gel3. used frequently for large DNA molecules

Acrylamide 1. a white odorless crystalline solid chemical compound2. soluble in water, ethanol, ether, chloroform3. used to synthesize poly-acrylamide which find many uses as water soluble thickeners

Starch Cellulose acetate

Types of matrix (supporting media)

DNA Agarose GelAn analytical technique used to separate DNA

by size1. Electric field induces

DNA to migrate toward the anode due to the net negative charge of the sugar phosphate backbone of the DNA

2. Longer molecules migrate more slowly

3. Visualized using a fluorescence dye special for DNA such as ethidium bromide

acrylamide polymer very stable gel can be made at a wide variety of concentrations large variety of pore sizes (powerful sieving

effect)

Polyacrylamide Gels

Sodium Dodecyl Sulfate = Sodium Lauryl Sulfate: CH3(CH2)11SO3

- Na+

Amphipathic molecule

Strong detergent to denature proteins

Binding ratio: 1.4 g SDS/g 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

Blotting and HybridizationBlotting and Hybridization

BlottingBlottingTransfer the DNA from the gel to a solid

supportTransferring of DNA, RNA, Protein to an

immobilizing binding matrix such as nitrocellulose paper or nylon

Northern blot(RNA)

Western blot(Protein)

Eastern blot(???)

Southern blot(DNA)

Blotting• Two methods :

– Capillary transfer– Electrophoretic transfer

SOUTHERN BLOTTING The technique was developed by E.M. Southern

in 1975. The Southern blot is used to detect the presence

of a particular piece of DNA in a sample. The DNA detected can be a single gene, or it

can be part of a larger piece of DNA such as a viral genome

The key to this method is hybridization. Hybridization-process of forming a double-

stranded DNA molecule between a single-stranded DNA probe and a single-stranded

target patient DNA.

SOUTHERN BLOTTINGThere are 2 important features of

hybridization:• The reactions are specific

The probes will only bind to targets with a complementary sequence.

• The probe can find one molecule of target in a mixture of millions of related but non-complementary molecules.

Southerns Blotting (DNA Blotting)

DNA fragments created by restriction digestion are separated on an agarose gel

Separated fragments are denatured and transferred to a membrane (blot) by blotting

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)

Southern blotting

Some Applications of DNA Blots Map restrictions sites near a particular locus

for gene isolation or allele analysis (e.g., RFLP restriction fragment length polymorphism)

Identity of closely related genes Confirmation of gene transfer or gene

disruption Detection of foreign DNA

RNA Blotting (Northern)

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

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

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

Use DNA to prove RNA

RNA Blotting (Northern)RNA Mixture

RNA

RNA

RNA Blotting (Northern)Advantage:- Very sensitive- Blots are reusable - Technical protocol is relatively simple- Can detect mRNA splice variants

Disadvantage:- Use of radioactivity (although non-radioactive techniques are available)- Laborious if many genes need to be tested- Assay is time-consuming

Applications of RNA BlotsDetect the expression level and transcript Detect the expression level and transcript size of a specific gene in a specific tissue or size of a specific gene in a specific tissue or at a specific time. Sometimes mutations do at a specific time. Sometimes mutations do

not affect coding regions but not affect coding regions but transcriptional regulatory sequences (e.g., transcriptional regulatory sequences (e.g.,

UAS/URS, promoter, splice sites, copy UAS/URS, promoter, splice sites, copy number, transcript stability)number, transcript stability)

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

HybridizationHybridization Pairing of complementary DNA and/or RNA

and/or protein

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

In-situ hybridisation

Chromosome in-situ hybridisation DNA probe detects sequences in chromosomes Map gene sequences

Tissue in-situ hybridisation RNA probe detects sequences in cells and

tissues Identify sites of gene expression Analyse tissue distribution of expression

Hybridization which is performed by denaturing the DNA of cell squash on a microscope slide so that reaction is possible with an added of probe

Oligonucleotide probes Single stranded DNA (usually 15-40 bp) Degenerate oligo-nucleotide 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)

Polymerase Chain ReactionPolymerase Chain Reaction (PCR)(PCR)

Polymerase Chain Reaction

Powerful technique for amplifying DNA

Amplified DNA are then separated by gel electrophoresis

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 Separation

Double 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) G/C content and Polypyrimidine (T, C)

or polypurine (A, G) stretches or 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/RNA Thermo-stable DNA polymerase e.g. Taq polymerase Oligonucleotides (primer) Design them well! Buffer Tris-HCl (pH 7.6-8.0)

Mg2+

dNTPs (dATP, dCTP, dGTP, dTTP)

OPTIMISING PCRTHE REACTION COMPONENTS

Organims, Organ, Tissue, cells (hair root, callus, leaves, root, seed)

Obtain the best starting material.Some can contain inhibitors of PCR, so they must

be removed e.g. Haem in bloodGood 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 40-50kbWhat 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°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 tubeAlways 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 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., 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 detection

The basis for many techniques to detect gene mutations The basis for many techniques to detect gene mutations (sequencing) - 1/6 X 10(sequencing) - 1/6 X 10-9-9 bp 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 controlDNA sequencing

Applications of PCR

DNA SequencingDNA Sequencing

DNA sequencingDNA sequencing Determination of nucleotide sequence

the determination of the precise sequence of nucleotides 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, T) Pyrimidines (C, T) are damaged by are damaged by hydrazinehydrazine

•Piperidine cleaves Piperidine cleaves the backbonethe backbone

•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 dideoxynucleotides DNA synthesis using deoxy- and dideoxynucleotides that results in termination of synthesis at specific that results in termination of synthesis at specific

nucleotidesnucleotides 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

ddATPGGCA

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