MOLECULAR DIAGNOSTICS N7-2006 L. Duroux Slides assembled from diverse sources.

MOLECULAR MOLECULAR DIAGNOSTICSDIAGNOSTICS

N7-2006N7-2006L. DurouxL. Duroux

Slides assembled from diverse sourcesSlides assembled from diverse sources

Recommended reading list - Recommended reading list - textbookstextbooks

Molecular Diagnostics: A Training and Study Molecular Diagnostics: A Training and Study GuideGuideGregory J. Tsongalis & William B. ColemanGregory J. Tsongalis & William B. Coleman

• Amer. Assoc. for Clinical Chemistry Amer. Assoc. for Clinical Chemistry , ISBN , ISBN 189088376X 189088376X

Diagnostic Molecular Microbiology: Diagnostic Molecular Microbiology: Principles and ApplicationsPrinciples and ApplicationsThomas F. Smith, Fred C. Tenover, Thomas J. White & Thomas F. Smith, Fred C. Tenover, Thomas J. White &

David H. PersingDavid H. Persing • ASM PressASM Press, ISBN , ISBN 155581056X155581056X

JournalsJournals

Journal of Journal of Molecular DiagnosticsMolecular Diagnostics http://jmd.amjpathol.org/http://jmd.amjpathol.org/

Nature BiotechnologyNature Biotechnology http://www.nature.com/nbt/http://www.nature.com/nbt/

Lecture PlanLecture Plan

1.1. Introduction: Definitions, Problematics, Introduction: Definitions, Problematics, ExamplesExamples

2.2. Immunological Diagnostic MethodsImmunological Diagnostic Methods

3.3. DNA Diagnostics MethodsDNA Diagnostics Methods

4.4. Bacterial BiosensorsBacterial Biosensors

1. INTRODUCTION1. INTRODUCTION

Molecular DiagnosticsMolecular Diagnostics

The success of modern medicine depends The success of modern medicine depends on the detection of specific molecules e.g.on the detection of specific molecules e.g.

VirusesViruses BacteriaBacteria FungiFungi ParasitesParasites ProteinsProteins In water, plants, soil and humans.In water, plants, soil and humans.

Molecular Diagnostics are Transforming Medicine

Pre-natal testing

Disease predisposition

Disease detection

Drug selection

Recurrence monitoring

Key questions

Need for Molecular tests

“Is the baby healthy? “

“What diseases is this patient at risk for?”

“Do this patient have disease?”

“What drugs should I prescribe?”

“How the disease returned?”

Molecular diagnostics is

>$3 billion market WW and

growing at >20% annually

Characteristics of a Detection SystemCharacteristics of a Detection System

A good detection system should have 3 qualities:A good detection system should have 3 qualities:♣ SensitivitySensitivity♣ SpecificitySpecificity♣ SimplicitySimplicity

SensitivitySensitivity means that the test must be able to means that the test must be able to detect very small amounts of targetdetect very small amounts of target even in the even in the presence of other molecules.presence of other molecules.

SpecificitySpecificity: the test yields a : the test yields a positive result for positive result for the target molecule only.the target molecule only.

SimplicitySimplicity: the test must be able to : the test must be able to run run efficiently and inexpensivelyefficiently and inexpensively on a routine basis. on a routine basis.

MassARRAY Diagnostics Are Being MassARRAY Diagnostics Are Being Developed For Multiple Disease AreasDeveloped For Multiple Disease Areas

PrenatalDiagnostics

Oncology

InfectiousDisease

TransplantationMedicine

Genetic Testing • High throughput testing for genetic disorders including SNPs, insertions, deletions

• Examples: Factor II, Factor V, CFTR

• Non-invasive detection of fetal diseases • Examples: Down syndrome, RhD, cystic

fibrosis

• Early diagnosis of cancer• Example: circulating tumor DNA

• Non-invasive, early detection of organ rejection

• Example: urine testing for kidney rejection

• Pathogen identification and early detection• Examples: identification of multi drug

resistant mycobacteria, early detection of drug-resistant viral strains, e.g. HIV, HBV, HCV

• Progress is being made in all of these areas

• Each of these areas are commercially attractive

• In some cases, the MassARRAY platform is uniquely qualified for specific tests

• More tests will be added to the platform as these tests are rolled out

Example 1Example 1

Pre-natal DiagnosticsPre-natal Diagnostics

Non-invasive Pre-Natal Diagnostics: Non-invasive Pre-Natal Diagnostics: A Large, Untapped Market A Large, Untapped Market

130 million live births worldwide per year130 million live births worldwide per year– 8 million live births in US and Europe 8 million live births in US and Europe

per yearper year

6% of all babies are born with birth defects6% of all babies are born with birth defects– over 900 fetal genetic disordersover 900 fetal genetic disorders

Down syndrome is the most common Down syndrome is the most common chromosomal abnormality chromosomal abnormality

– Although risk increases with age, 80% Although risk increases with age, 80% of Down of Down births are in women <35 births are in women <35 years oldyears old

– Even though limited to high risk Even though limited to high risk mothers, mothers, amniocentesis is a $600 amniocentesis is a $600 million market in US million market in US and $1.5 billion and $1.5 billion market worldwidemarket worldwide

Although market is large and there is unmet need, there are no accurate,

non-invasive prenatal diagnostic

tests (“NIPD”) available

Non-Invasive Prenatal TestingNon-Invasive Prenatal Testing

Platform independentPlatform independent Mass spec, RT-PCR, othersMass spec, RT-PCR, others Covers all prenatal tests Covers all prenatal tests RhD – RhD – Commercialize homebrew in 1H07 Commercialize homebrew in 1H07 HemoglobinopathiesHemoglobinopathies

Thalassemias – Asian market Thalassemias – Asian market Cystic fibrosisCystic fibrosis OthersOthers With advanced pre-analyticsWith advanced pre-analytics Aneuploidies – Down SyndromeAneuploidies – Down Syndrome

Example 2Example 2

Genetic testing Genetic testing

0

200

400

600

800

1000

1200

1400

1600

1800

1997 1998 1999 2000 2001 2002 2003

www.ncbi. nlm.nih.govwww.genetests.org

Molecular Genetic Testing

Disease Genes Identified

Molecular Tests Offered in North America

Molecular Tests Offered In Ontario

Types of Mutations Tested

Disease

Point mutations?

Deletions &duplications?

Few recurrentmutations?

Many uniquemutations?

Also with pointmutations?

Whole gene?Some exons?

Other mutations?

Diagnosis of Batten Disease:

Neuronal Ceroid Lipofuscinoses

Batten disease is a fatal, inherited disorder of the nervous system that begins in childhood. It is the buildup of lipopigments in the body's tissues. Lipopigments are made up of fats and proteins.

Normal

MutantC451T

Primer Extension Test for NCL Mutations

An enzyme called palmitoyl-protein thioesterase has been shown to be insufficiently active in the infantile form of Batten disease. This condition is now referred to as NCL1.

Normal

C70G IVS

delAT

1.02del

C622T

C451T

A223C

A364T

G284V

Multiplexed Analysis of 9 NCL Mutations

NCL2: late infantile form, mutations of an acid protease.NCL3: juvenile form, gene not been identified

2- Immunological 2- Immunological Diagnostics MethodsDiagnostics Methods

Applications of Immunoassays Applications of Immunoassays

Analysis of hormones, vitamins, metabolites, Analysis of hormones, vitamins, metabolites, diagnostic markersdiagnostic markers Eg. ACTH, FSH, T3, T4, Glucagon, Insulin, Eg. ACTH, FSH, T3, T4, Glucagon, Insulin,

Testosterone, vitamin B12, prostaglandins, Testosterone, vitamin B12, prostaglandins, glucocorticoids, glucocorticoids,

Therapeutic drug monitoring: Therapeutic drug monitoring: Barbiturates, morphine, digoxin Barbiturates, morphine, digoxin

Diagnostic procedures for detecting infection Diagnostic procedures for detecting infection HIV, Hepatitis A, B, etc…HIV, Hepatitis A, B, etc…

Antigen-AntibodyAntigen-AntibodyInteractionsInteractions

- a bimolecular association - a bimolecular association

involving various non-covalent interactionsinvolving various non-covalent interactions- Is similar to an enzyme-substrate interactions, Is similar to an enzyme-substrate interactions,

but not lead to an irreversible chemical alterationbut not lead to an irreversible chemical alteration

1.1. Strength of Antigen-Antibody InteractionsStrength of Antigen-Antibody Interactions

2.2. Cross-ReactivityCross-Reactivity

3.3. Precipitation ReactionsPrecipitation Reactions

4.4. Agglutination ReactionsAgglutination Reactions

5.5. RadioimmunoassayRadioimmunoassay

6.6. Enzyme-Linked Immunosorbent AssayEnzyme-Linked Immunosorbent Assay

7.7. Western BlottingWestern Blotting

8.8. ImmunoprecipitationImmunoprecipitation

9.9. ImmunofluorescenceImmunofluorescence

10.10. Flow Cytometry and FluorescenceFlow Cytometry and Fluorescence

11.11. Alternatives to Antigen-Antibody ReactionsAlternatives to Antigen-Antibody Reactions

12.12. Immunoelectron MicroscopyImmunoelectron Microscopy

Strength of Ag-Ab InteractionsStrength of Ag-Ab Interactions

Antibody affinity- is a quantitative measure of binding strength- combined strength of the noncovalent interactions between a binding site on an Ab & monovalent Ag

Antibody avidity- Incorporates affinity of multiple binding sites- True strength of the Ab-Ag interaction within biological systems- The interaction at one site will increase the possibility of reaction at a second site- High avidity can compensate for low affinity ( secreted pentameric IgM has a higher avidity than IgG )

Strength of Ag-Ab InteractionsStrength of Ag-Ab Interactions

Forward & reverse rate constants ( k1 & k-1)Association & dissociation constants ( Ka & Kd ) for 3 ligand-Ab interaction

- High affinity complexes have high Ka values- Very stable complexes have very low values of Kd

Sensitivity of various immunoassays

CROSS-REACTIVITY

(i) Cowpox antigens in vaccinia virus are cross-reactive to smallpox antigens in variola virus ( share similar or identical epitope )∵

(ii) Rabies & JE vaccine >>> encephalitis ( 뇌염 ) (: rabbit brain antigen contaminated vs human brain Ag )

(iii) Streptococcus pyogenes infection >>> heart & Kidney damage following infection (: cell wall proteins called M antigens vs Myocardial & skeletal muscle proteins ).

(iv) Original antigenic sin. - The existence of long-lived lymphocytes & crossreactivity - Vaccination with one strain of flu elicited Ab responses to another flu strain. (v) cross-reacting bacterial Ag vs glycoproteins on RBC)

- Antibody elicited by one Ag can cross-react with unrelated Ag.- occurs if two different Ags share identical or very similar epitope

ABO blood types

- The antibodies are induced by exposure to cross-reacting microbial antigens present on common intestine bacteria.- ABO blood-group antigens have subtle differences in the terminal residues of the sugars on glyco-proteins in RBC.- Providing the basis for blood typing test in blood transfusion

2. Cross-Reactivity2. Cross-Reactivity

-The original home pregnancy test kit employed hapten inhibition (agglutination inhibition) to determine the presence or absence of human chorionic gonadotropin (HCG) >>> The kits currently on the market use ELISA-based assays.-Also used to determine the use of illegal drugs, & immunity (Ab) to virus (rubella).

Agglutination ReactionsAgglutination Reactions

ELISAELISA

Addition of a Addition of a specific antibodyspecific antibody (primary (primary antibody) which will bind to the test molecule if it antibody) which will bind to the test molecule if it is present.is present.

WashingWashing to remove unbound molecules. to remove unbound molecules. Addition of Addition of secondary antibodysecondary antibody which will bind which will bind

to the primary antibody.to the primary antibody. The secondary antibody usually has attached to The secondary antibody usually has attached to

it an it an enzymeenzyme e.g. e.g. alkaline phosphatasealkaline phosphatase.. WashWash to remove unbound antibody. to remove unbound antibody. Addition of a Addition of a colourless substratecolourless substrate which will which will

react with the secondary antibody to give a react with the secondary antibody to give a colour reactioncolour reaction which indicates a positive result. which indicates a positive result.

ELISAELISAOne of many variants One of many variants

of the techniqueof the technique

http://www.immunospot.comhttp://www.immunospot.com/elisa-animation.html/elisa-animation.html

Advantages of ELISAAdvantages of ELISA Sensitive: nanogram levels or lowerSensitive: nanogram levels or lower ReproducibleReproducible Minimal reagentsMinimal reagents Qualitative & Quantitative Qualitative & Quantitative

Qualitative Qualitative Eg HIV testing Eg HIV testing quantitative assays quantitative assays Eg Ther. Drug Monitoring Eg Ther. Drug Monitoring

Greater scope : Wells can be coated with Greater scope : Wells can be coated with Antigens OR Antibodies Antigens OR Antibodies

Suitable for automation Suitable for automation high speedhigh speed NO radiation hazardsNO radiation hazards

Detection based on enzyme catalyzed reactions (alkaline , ⓟ horseradish peroxidase, & β-galactosidase) : safer & less costly.

to detect Ab (HIV)

to detect Ag

to detect Ag

ELISA: ELISA:

ManyMany

variants variants

The ELISPOT assay, a modification of the ELISA assay to determine quantitatively the # of cells in a population that are producing specific Ab or cytokine.

6. ELISA 6. ELISA

-> precipitates & forms a spot only on the areas of the well where cytokine-secreting cells had been deposited.

Western blotting

: separates the components according to their molecular weight.

: the proteins in the gel are transferred to the sheet of nitrocellulose or nylon by the passage of an electric current.

: probed with Ab & then radiolabeled or enzyme-linked 2nd Ab.

: a position is visualized by means of an ELISA reaction.

Immuno-precipitates can be collected using magnetic beads coupled to a secondary antibody.

Immunoprecipitation- Ag-Ab attached to a synthetic bead complex >>> 0- labeling Ag with radiolabeled leucine, cysteine, or methionine → A radiolabeled Ag-Ab complex → 0 → SDS•PAGE → autoradiography- Ag-Ab complex + 2nd Ab attatched to a synthetic bead or magnetic beads >>> 0 or magnet

EM showing a cell with magnetic beads attached to its surface via antibodies.

Immunofluorescence

mIgM-producing B cells indirectly stained with rhodamine-conjurated secondary Ab under a fluorescence microscope.

Fluorochromes-Fluorescein (490→517nm)-Rhodamine (515→546nm)-Phycoerythrin : absorb light of one wavelength & emit fluorescence at a longer wavelength than fluorescein.

An immunoelectronmicrograph of the surface of a B-cell lymphoma was stained with two antibodies (Ab against class II MHC labeled with 30nm gold particles, & another Ab against class I MHC w/ 15nm gold particles.(The density of class I exceeds that of class II)- Electron-dense label (ferritin or colloidal gold) is conjugated to the Fc portion.

electron-dense labelsabsorb electrons.

Immuno Electron Microscopy

Alternatives to Ag-Ab Reactions

Ag-Ab-Ab*→Ag-IgG-A/G* or Ag-Ab-biotin-(a)vidin*

① Protein A (from staphylococcus) & protein G (from streptococcus) - bind to rhe Fc region of lgG molecules (ka ～ 108) - used to detect lgG molecules in the Ag-Ab complexes - used to isolate lgG molecules in the affinity columns

② Avidin (from egg whites) & streptavidin (from streptomyces avidinii) conjugated with an enzyme, fluorochrome, radioactive label) - bind to biotin (a vitamin) with higher affinity (ka ～ 1015) - Ab can be labeled with (ka ～ 1018)

3. DNA Diagnostic 3. DNA Diagnostic SystemsSystems

Problematics & Problematics & SolutionsSolutions

Genetic testing in individuals Genetic testing in individuals and populationsand populations

Does THIS patient have ANY mutation in ANY Does THIS patient have ANY mutation in ANY gene that would explain his disease? gene that would explain his disease?

Does THIS patient have ANY mutation in THIS Does THIS patient have ANY mutation in THIS gene that might cause his disease? gene that might cause his disease?

Does THIS patient have a 3-bp deletion of Phe Does THIS patient have a 3-bp deletion of Phe codon in CFTR gene? codon in CFTR gene?

NOT POSSIBLE TO SAY

NEED LOTS OF EFFORTS TO ANSWER

THAT IS A RIGHT QUESTION !!!

The choice of material to test The choice of material to test

DNADNA most common; tested by PCRmost common; tested by PCR

Sometimes tested by Southern blottingSometimes tested by Southern blotting

RNARNA RT-PCR allow to test genes RT-PCR allow to test genes directly, without breaking them into exons. directly, without breaking them into exons.

Allow to detect alternative spliced isoforms.Allow to detect alternative spliced isoforms. Allow to test for unknown mutations faster Allow to test for unknown mutations faster

than in DNAthan in DNA

How to obtain DNA specimenHow to obtain DNA specimen

Blood sampleBlood sample (most common for adult testing); (most common for adult testing); Mouthwashes or buccal scrapesMouthwashes or buccal scrapes (non-invasive); (non-invasive); Chorionic villusChorionic villus biopsy samples biopsy samples (fetal DNA); (fetal DNA); Hair, semenHair, semen (criminology) (criminology) One or two cells removed from 8-cell embryo One or two cells removed from 8-cell embryo

(in vitro fertilisation) Archived pathological specimensArchived pathological specimens (typing dead (typing dead

peoples, tumor samples in paraffin blocks);peoples, tumor samples in paraffin blocks); Paper cardsPaper cards with blood drops on them with blood drops on them

Methods of mutation scanning Methods of mutation scanning (when we do not know where is our mutation) (when we do not know where is our mutation)

SequencingSequencing -- most direct method; -- most direct method; DetectingDetecting mismatchesmismatches or heteroduplex DNA or heteroduplex DNA

molecules; molecules; Single-strandSingle-strand conformational analysis; conformational analysis; Protein truncation testProtein truncation test (PTT); (PTT); Detecting of Detecting of deletionsdeletions; ; Detection of Detection of methylationmethylation

DNA Diagnostic SystemsDNA Diagnostic Systems

DNA Diagnostic Systems include:DNA Diagnostic Systems include: DNA HybridizationDNA Hybridization DNA SequencingDNA Sequencing PCRPCR Restriction endonuclease analysisRestriction endonuclease analysis RAPD (random amplified polymorphic RAPD (random amplified polymorphic

DNA)DNA) DNA fingerprintingDNA fingerprinting

Hybridization methodsHybridization methods

DNA HybridizationDNA Hybridization Bacterial and viral pathogens may be Bacterial and viral pathogens may be

pathogenic because of the presence of pathogenic because of the presence of specific genesspecific genes or sets of genes. or sets of genes.

Genetic diseases often are due toGenetic diseases often are due to mutations mutations or or absenceabsence of particular gene or genes. of particular gene or genes.

These genes (DNA) can be used as These genes (DNA) can be used as diagnostic tools.diagnostic tools.

This involves using a This involves using a DNA probeDNA probe during during DNA hybridizationDNA hybridization..

What is a DNA probe?What is a DNA probe? How does DNA hybridization work?How does DNA hybridization work?

DNA HybridizationDNA Hybridization

For DNA hybridization:For DNA hybridization: A A probeprobe is needed which will anneal to the target is needed which will anneal to the target

nucleic acid.nucleic acid. Attach the targetAttach the target to a solid matrix e.g. membrane. to a solid matrix e.g. membrane. DenaturationDenaturation of of both the probe and target.both the probe and target. Add the denatured probe in a solution to the target.Add the denatured probe in a solution to the target. If there is If there is sequence homologysequence homology between the target between the target

and the probe, the probe will hybridize or anneal to and the probe, the probe will hybridize or anneal to the target. the target.

DetectionDetection of the hybridized probe e.g. by of the hybridized probe e.g. by autoradiography, chemiluminsence or colorimetric.autoradiography, chemiluminsence or colorimetric.

DNA hybridization movieDNA hybridization movie

At At http://www.imagecyte.com/http://www.imagecyte.com/

Example: Detection of MalariaExample: Detection of Malaria

Malaria is caused by the parasite Malaria is caused by the parasite Plasmodium Plasmodium falciparumfalciparum..

The parasite infects and destroys The parasite infects and destroys red bloodred blood cells. cells. Symptoms include fever, rashes and damage to Symptoms include fever, rashes and damage to

brain, kidney and other organs.brain, kidney and other organs. Current treatment involves Current treatment involves microscopic microscopic

observationsobservations of blood smears, which is labour of blood smears, which is labour intensive.intensive.

Other methods e.g Other methods e.g ELISA ELISA does not differentiate does not differentiate between past and present infection.between past and present infection.

Why?Why?

Detection of MalariaDetection of Malaria

A DNA diagnostic system would only measure A DNA diagnostic system would only measure current infectioncurrent infection. . (Why?)(Why?)

The procedure involves:The procedure involves: A A genomic librarygenomic library of the parasite was of the parasite was

screened with probes for parasitic DNA.screened with probes for parasitic DNA. The probes which The probes which hybridized stronglyhybridized strongly

(highly repetitive DNA) were tested further.(highly repetitive DNA) were tested further. The probes were tested for their ability to The probes were tested for their ability to

hybridize to other hybridize to other PlasmodiumPlasmodium species species which do not cause malaria and to which do not cause malaria and to human human DNADNA..

DAYS POST ONSETDAYS POST ONSET

1 2 3 4 5 6 7 8 9 10 -14 to -2 0

IgMIgG

ELISAELISAP/NP/N#pfu/ml#pfu/ml

Malaria

250

illnessillness

Theoretical Depiction of Malaria InfectionTheoretical Depiction of Malaria Infection & Immune Response& Immune Response

2

20

Serology AssaysMicrobe Assays

Neutralizing Ab

Detection of MalariaDetection of Malaria

Probes which hybridized to Probes which hybridized to P. falciparumP. falciparum only could be used as a diagnostic tool.only could be used as a diagnostic tool.

The probe was able to detect 10 pg of The probe was able to detect 10 pg of purified DNA or 1 ng of DNA in blood smear.purified DNA or 1 ng of DNA in blood smear.

Other DNA probes were developed for the Other DNA probes were developed for the following diseases:following diseases:

Salmonella typhiSalmonella typhi (food poisoning) (food poisoning) E. coliE. coli (gastroenteritis) (gastroenteritis) Trypanosoma cruziTrypanosoma cruzi (chagas’ disease) (chagas’ disease)

TaqManTaqMan®® Probes Probes

Unbound probe free in solution

Probe and primer bind target, FRET occurs

Light Emission

Donor dye (Reporter) Acceptor dye

(Quencher)

Light

Energy transfer

Taq

Taq extends and hydrolyzes probe, donor dye free to emit fluorescence --> accumulation of signal

Taq

Light EmissionLight

Taqman Probe designTaqman Probe design

20-30 bp in length, Tm 10°C higher than primers.20-30 bp in length, Tm 10°C higher than primers. 35-65% G/C; more Cs than G’s. Can try as high as 80% or 35-65% G/C; more Cs than G’s. Can try as high as 80% or

as low as 20% if the region is particularly GC or AT rich.as low as 20% if the region is particularly GC or AT rich. Avoid runs of 3+ of the same nucleotide, especially G’s.Avoid runs of 3+ of the same nucleotide, especially G’s. 5’ base 5’ base G. G. When the probe and primers anneal to the target, the 5’ end When the probe and primers anneal to the target, the 5’ end

of probe should be 3 nucleotides from the 3’ end of the of probe should be 3 nucleotides from the 3’ end of the primer on same strand (max of 10-12).primer on same strand (max of 10-12).

Test that primers and probe are not complementary to each Test that primers and probe are not complementary to each other. (delta G free energy at 25C should be greater than -2)other. (delta G free energy at 25C should be greater than -2)

Molecular BeaconsMolecular Beacons

Probe hybridized to DNA template

LightLight Emission

Probe in preferred closed structure

DNA template

Reporter dye Quencher

Stem

Loop

Light

Grd. St. Quenching

Molecular Beacon designMolecular Beacon design

Begin design as for a hydrolysis probe.Begin design as for a hydrolysis probe. Tm of probe region should be 7-10°C above target annealing temp.Tm of probe region should be 7-10°C above target annealing temp. To the chosen sequence add a stemTo the chosen sequence add a stem 5-7 bp in length, with similar Tm 5-7 bp in length, with similar Tm as the probe region.as the probe region. Test folding using appropriate software (Test folding using appropriate software (mfoldmfold).).

http://www.bioinfo.rpi.edu/applications/mfold/http://www.bioinfo.rpi.edu/applications/mfold/ Check that there is no complementarity between primers and probe.Check that there is no complementarity between primers and probe. Tm of probe alone and probe + complement should be verified Tm of probe alone and probe + complement should be verified

experimentallyexperimentally

Molecular Beacon Melting CurveMolecular Beacon Melting Curve

Properly Properly designed designed Molecular Molecular Beacons can Beacons can effectively effectively discriminate discriminate between between targets with a targets with a single bp single bp mismatch.mismatch.

Beacon + Target

Beacon + 1bp

mismatched

target

Beacon alone

FISH DiagnosisFISH Diagnosis

Analyse chromosomesAnalyse chromosomes

Sexing for X-linked diseaseSexing for X-linked disease

Chromosome abnormalitiesChromosome abnormalities

Age related aneuoploidyAge related aneuoploidy

Genetic AnalysisGenetic Analysis

Prenatal DiagnosisPrenatal Diagnosis

Chorionic villusChorionic villus sampling (CVS)sampling (CVS)

• 10-14wks10-14wks AmniocentesisAmniocentesis

• (15-16wks)(15-16wks) Fetal blood samplingFetal blood sampling

(FBS)(FBS)

Preimplantation Preimplantation Genetic DiagnosisGenetic Diagnosis

early embryos early embryos • IVF IVF • selection for transfer to selection for transfer to

the uterus the uterus

Inherited disordersInherited disorders

Couple with a genetic disorderCouple with a genetic disorder

OptionsOptions Reproductive rouletteReproductive roulette No more childrenNo more children AdoptionAdoption Gamete donationGamete donation Prenatal diagnosis / Prenatal diagnosis /

terminationtermination PGDPGD

AffectedAffected ChildrenChildren Other family membersOther family members

Cleavage Stage BiopsyCleavage Stage Biopsy

Sexing Embryos for PDG:Sexing Embryos for PDG: FISH analysis of interphase FISH analysis of interphase

nucleinucleiChromosome XChromosome X Chromosome YChromosome Y Chromosome 16Chromosome 16

Normal FemaleNormal Female Normal MaleNormal Male

Chromosome AbnormalitiesChromosome Abnormalities

TranslocationsTranslocations RobertsonianRobertsonian

• Only 13,14,15,21,22Only 13,14,15,21,22 ReciprocalReciprocal

InsertionsInsertions InversionsInversions Ring ChromosomesRing Chromosomes

PGD of Chromosome Abnormalities: PGD of Chromosome Abnormalities: Robertsonian Translocation Carrier 45,XY,der(13q;14q)(q10;q10)Robertsonian Translocation Carrier 45,XY,der(13q;14q)(q10;q10)

Chromosome 13Chromosome 13 Chromosome 14Chromosome 14

Normal for Chromosomes 13 & 14Normal for Chromosomes 13 & 14 Monosomy Monosomy 1414

Preimplantation Genetic Diagnosis of Chromosomal Preimplantation Genetic Diagnosis of Chromosomal Imbalance for Reciprocal Translocation Carriers using Imbalance for Reciprocal Translocation Carriers using

Triple-colour FISHTriple-colour FISH

Reciprocal Translocation 46,XX,t(5;9)(q32;p13)Reciprocal Translocation 46,XX,t(5;9)(q32;p13)

5q325q32

9p139p13

5 der 5 der 9 95 der 5 der 9 9

Aneuploidy ScreeningAneuploidy Screening

Older women likely to produce abnormal oocytesOlder women likely to produce abnormal oocytes Leads to chromosomally abnormal embryosLeads to chromosomally abnormal embryos

increase in miscarriageincrease in miscarriage lower pregnancy ratelower pregnancy rate

Chromosomes commonly involvedChromosomes commonly involved 13, 16, 18, 21, X and Y13, 16, 18, 21, X and Y

Used for older women withUsed for older women with recurrent IVF failurerecurrent IVF failure recurrent miscarriagerecurrent miscarriage

Chromosomes in human Chromosomes in human embryosembryos

NORMALNORMAL All cells uniformly diploidAll cells uniformly diploid

ABNORMALABNORMAL All cells uniformly abnormal eg trisomy 21All cells uniformly abnormal eg trisomy 21

MOSAICMOSAIC Two or more cell lines presentTwo or more cell lines present

• often diploid with aneuploid or tetraploid cellsoften diploid with aneuploid or tetraploid cells

CHAOTICCHAOTIC Different chromosome pattern in every cellDifferent chromosome pattern in every cell

Sequencing methodsSequencing methods

Sequencing Sequencing (cost – DKK 50,00 per run)(cost – DKK 50,00 per run)

As sequencing becomes more and more cheap, As sequencing becomes more and more cheap,

it pushes other methods backward. it pushes other methods backward.

For sequencing of genomic DNA, For sequencing of genomic DNA,

every exon is amplified separatelyevery exon is amplified separately (Typical sequencing run – 500bp; typical exon size – 145 bp)(Typical sequencing run – 500bp; typical exon size – 145 bp)

Example of Diagnostic for

Duchenne Muscular Dystrophy (DMD)

X-linked and affect mainly X-linked and affect mainly males an estimated 1 in 3500 males an estimated 1 in 3500 boys worldwideboys worldwide

DMD encodes a large DMD encodes a large structural protein: dystrophinstructural protein: dystrophin

strengthen muscle cells by strengthen muscle cells by anchoring elements of the anchoring elements of the internal cytoskeleton to the internal cytoskeleton to the surface membranesurface membrane

Mutated dystrophin leads to Mutated dystrophin leads to ”implosion” of muscle cells”implosion” of muscle cells

60

40

20

Deletion Duplication Point

%

DMD Mutation Types

DNA Sequencing

Normal

Carrier

Minisequencing by primer extension Minisequencing by primer extension

DNA polymerase + one of the four labeled dNTPs = sequencing of one nucleotide

http://las.perkinelmer.com/content/snps/protocol.asp

+ HPLC analysis

Principle of PyrosequencingPrinciple of Pyrosequencing

Step 1A sequencing primer is hybridized to a single stranded, PCR amplified DNA template, and incubated with the enzymes:-- DNA polymerase, -- ATP sulfurylase, -- luciferase -- apyrase,

and the substrates:-- adenosine 5´ phosphosulfate (APS) -- luciferin.

http://www.pyrosequencing.com/pages/technology.html

Principle of PyrosequencingPrinciple of Pyrosequencing

Step 2Step 2The first of four dNTP is added to the reaction. The first of four dNTP is added to the reaction.

DNA polymerase catalyzes the incorporation of the DNA polymerase catalyzes the incorporation of the dNTP into the DNA strand, if it is complementary to dNTP into the DNA strand, if it is complementary to the base in the template strand. the base in the template strand.

Each incorporation event is accompanied by release Each incorporation event is accompanied by release of an equimolar quantity of pyrophosphate (PPi)of an equimolar quantity of pyrophosphate (PPi)

Principle of PyrosequencingPrinciple of Pyrosequencing

Step 3ATP sulfurylase PPi ATP. ATP used in the conversion of luciferin to oxyluciferin (visible light proportional to ATP production). The light is detected by a charge coupled device (CCD) camera and seen as a peak in a pyrogram™.

Light signal is proportional to the number of nucleotides incorporated.

Step 4Step 4ApyraseApyrase,, a nucleotide degrading enzyme, a nucleotide degrading enzyme, continuously degrades unincorporated dNTPs and continuously degrades unincorporated dNTPs and excess ATP. When degradation is complete, another excess ATP. When degradation is complete, another dNTP is added. dNTP is added.

Principle of PyrosequencingPrinciple of Pyrosequencing

Principle of PyrosequencingPrinciple of Pyrosequencing

Addition of dNTPs is performed one at a time.

thio triphosphate (dATP-alphaS)is used as a substitute for the natural (dATP), as natural dATP is not good for luciferase

Problems arising in mutation scanningProblems arising in mutation scanning

Example: Example:

Duchenne muscular dystrophyDuchenne muscular dystrophyProblems: 1. Gene is large, 2,4 Mb, 79 exonsHard to find point mutation

2. High Frequency of new mutations

(30% of cases);

3. First mutation carrier is often a mosaic(blood may be not a mutation carrier)

DNA Polymerase-DNA Polymerase-based Diagnosticsbased Diagnostics

Polymerase Chain ReactionPolymerase Chain Reaction

PCR uses 2 sequence PCR uses 2 sequence specific specific oligonucleotide primersoligonucleotide primers to amplify the to amplify the target DNA.target DNA.

The presence of the appropriate The presence of the appropriate amplified amplified size fragmentsize fragment confirms the presence of the confirms the presence of the target.target.

Specific primers are now available for the Specific primers are now available for the detection of many pathogens including detection of many pathogens including bacteria (bacteria (E. coliE. coli, , M. tuberculosisM. tuberculosis), viruses ), viruses (HIV) and fungi. (HIV) and fungi.

Using PCR to Detect for HIVUsing PCR to Detect for HIV RT-PCRRT-PCR (reverse transcriptase PCR). (reverse transcriptase PCR). HIV has a ssRNA genome.HIV has a ssRNA genome. Lyse plasma cells from the potentially Lyse plasma cells from the potentially

infected person to release HIV RNA infected person to release HIV RNA genome.genome.

The RNA is precipitated using isopropanol.The RNA is precipitated using isopropanol. Reverse transciptase is used to make a Reverse transciptase is used to make a

cDNAcDNA copy of the RNA of the virus. copy of the RNA of the virus. This cDNA is used as a template to make This cDNA is used as a template to make

dsDNAdsDNA..

RT-PCR Diagnosis of HIVRT-PCR Diagnosis of HIV

Using PCR to Detect for HIVUsing PCR to Detect for HIV

Specific primers are used to amplify a 156 Specific primers are used to amplify a 156 bp portion of the HIVbp portion of the HIV gaggag gene gene..

Using standards the amount of PCR Using standards the amount of PCR product can be used to determine the product can be used to determine the viral viral loadload. .

PCR can also be used as a prognostic tool PCR can also be used as a prognostic tool to determine viral load.to determine viral load.

This method can also be used to This method can also be used to determine the effectiveness antiviral determine the effectiveness antiviral therapy.therapy.

60

40

20

Deletion Duplication Point

%

Detecting DMD Deletions by PCR

3'5'

1 10 20 30 40 50 60 70

Deletions in DMD Gene : dystrophin

Exons

BMD

DMD

Intermediate

DMD Deletion Carrier Analysis

by PCR/ size analysis

DNA Fingerprinting (RFLP)DNA Fingerprinting (RFLP)

RFLP = RFLP = Restriction Fragment Length PolymorphismRestriction Fragment Length Polymorphism

Regular fingerprinting analyses Regular fingerprinting analyses phenotypicphenotypic traits.traits.

DNA fingerprinting analyses DNA fingerprinting analyses genotypic genotypic traits.traits. DNA fingerprinting (DNA typing) is used to DNA fingerprinting (DNA typing) is used to

characterize biological samples e.g.characterize biological samples e.g. In legal proceedings to identify suspects In legal proceedings to identify suspects

and clear others.and clear others. Paternity testingPaternity testing

Restriction fragment length Restriction fragment length polymorphism (RFLP)polymorphism (RFLP)

Restriction fragment length polymorphism Restriction fragment length polymorphism (RFLP)(RFLP)

Very simple; Very simple; Not allow high-throughput detection; Not allow high-throughput detection; Even as many restriction enzymes are known,Even as many restriction enzymes are known,

some mutation sites do not correspond to anysome mutation sites do not correspond to any

Rare endonucleases are difficult to work with, and often of a poor quality

Restriction fragment length polymorphism Restriction fragment length polymorphism (RFLP)(RFLP)

Diagnostic restriction site could be

introduced artificiallyby purposedly mismatched

PCR primer

Diagnosis of sickle cell anemia by RFLP.Diagnosis of sickle cell anemia by RFLP.

Sickle cell anemia is a genetic disease which is Sickle cell anemia is a genetic disease which is caused by a single nucleotide change in the caused by a single nucleotide change in the 66thth aa aa of the of the chain of hemoglobin. chain of hemoglobin.

A (normal) A (normal) glutamic acidglutamic acid and S (sickle) and S (sickle) valine.valine. In the homozygous state SS the red blood cells In the homozygous state SS the red blood cells

are irregularly shaped.are irregularly shaped. The disease results in progressive anemia and The disease results in progressive anemia and

damage to heart, lung, brain, joints and other damage to heart, lung, brain, joints and other organ systems.organ systems.

This occurs because the mutant hemoglobin is This occurs because the mutant hemoglobin is unable to unable to carry enough oxygencarry enough oxygen to supply these to supply these systems.systems.

Diagnosis of Sickle Cell AnemiaDiagnosis of Sickle Cell Anemia

The single mutation in hemoglobin cause a The single mutation in hemoglobin cause a change in the restriction pattern of the change in the restriction pattern of the globin globin gene gene abolishing a abolishing a CvnCvnI siteI site..

CvnCvnII site CCsite CCTNAGG (N = any nt)TNAGG (N = any nt) Normal DNA sequence CCTGNormal DNA sequence CCTGAAGG (A)GG (A) Mutant DNA sequence CCTGMutant DNA sequence CCTGTTGG (S)GG (S) Two primers which flank the mutant region of the Two primers which flank the mutant region of the

globin gene is used during PCR to amplify this globin gene is used during PCR to amplify this region of the gene.region of the gene.

The PCR products is digested with The PCR products is digested with CvnCvnI and I and separated by agarose gel electrophoresis.separated by agarose gel electrophoresis.

Detection Detection of Sickle of Sickle

cell anemia cell anemia by by

PCR/RFLPPCR/RFLP

Random Amplified Polymorphic DNA (RAPD)Random Amplified Polymorphic DNA (RAPD)

Another method widely used in Another method widely used in characterization of DNA is characterization of DNA is RAPDRAPD..

RAPD is often used to RAPD is often used to show relatednessshow relatedness among DNA populations.among DNA populations.

In this procedure arbitrary (In this procedure arbitrary (randomrandom) ) primers are used during PCR to produce a primers are used during PCR to produce a fingerprint of the DNA.fingerprint of the DNA.

A single primer is used which must anneal A single primer is used which must anneal in 2 places on the DNA template and region in 2 places on the DNA template and region between the primers will be amplified.between the primers will be amplified.

Random Amplified Polymorphic DNA Random Amplified Polymorphic DNA (RAPD)(RAPD)

The primers (8-10nt) are likely to anneal in The primers (8-10nt) are likely to anneal in many many placesplaces on the template DNA and will produce a on the template DNA and will produce a variety of sizesvariety of sizes of amplified products. of amplified products.

Amplified products are separated by agarose gel Amplified products are separated by agarose gel electrophoresis and visualized.electrophoresis and visualized.

If the samples have similar genetic make up then the If the samples have similar genetic make up then the pattern of bands on the gel will be similar and vice pattern of bands on the gel will be similar and vice versa.versa.

This procedure is widely used to differentiate between This procedure is widely used to differentiate between different cultivars/varieties of the same plant.different cultivars/varieties of the same plant.

Issues to consider when using this procedure include Issues to consider when using this procedure include reproducibility, quality of DNA, and several primers reproducibility, quality of DNA, and several primers may have to be used.may have to be used.

RAPDRAPDfingerprintfingerprint

PCR/OLAPCR/OLAOligonucleotide Ligation AssayOligonucleotide Ligation Assay

Like sickle cell anemia many genetic Like sickle cell anemia many genetic diseases are caused by mutant genes.diseases are caused by mutant genes.

Many diseases are caused by a single Many diseases are caused by a single nucleotide (nt) change in the wild type nucleotide (nt) change in the wild type gene.gene.

A single nt change can be detected by A single nt change can be detected by PCR/OLAPCR/OLA

PCR/OLA PCR/OLA

PCR/OLAPCR/OLA

Bacterial BiosensorsBacterial Biosensors

Bacterial BiosensorsBacterial Biosensors Bacterial sensors can be used to test for Bacterial sensors can be used to test for

environmental pollutants.environmental pollutants. Bacteria with bioluminescent are good Bacteria with bioluminescent are good

candidates for pollutant sensors.candidates for pollutant sensors. In the presence of pollutants the In the presence of pollutants the

bioluminescent decreases.bioluminescent decreases. The structural genes (The structural genes (luxCDABDluxCDABD) encodes the ) encodes the

enzyme for bioluminescent was cloned into enzyme for bioluminescent was cloned into the soil bacteria the soil bacteria Pseudomonas fluorescensPseudomonas fluorescens..

The cells that luminescence to the greatest The cells that luminescence to the greatest extent and grew as well as the wild type were extent and grew as well as the wild type were tested as pollutant sensors.tested as pollutant sensors.

Bacterial BiosensorsBacterial Biosensors

To screen water samples for pollutants To screen water samples for pollutants (metal or organic) a suspension of (metal or organic) a suspension of P. P. fluorescens fluorescens was mixed with the solution to was mixed with the solution to be tested.be tested.

After a 15 min incubation the luminescence After a 15 min incubation the luminescence of the suspension was measured.of the suspension was measured.

When the solution contained low to moderate When the solution contained low to moderate levels of pollutants the bioluminescence was levels of pollutants the bioluminescence was inhibited.inhibited.

The procedure is rapid, simple, cheap and a The procedure is rapid, simple, cheap and a good screen for pollutants.good screen for pollutants.

Bacterial Bacterial BiosensorBiosensor