September 2005

Finding the needle in the haystack Finding the needle in the haystack LNA bases enhance SNP detection dramaticallyLNA bases enhance SNP detection dramatically

Olfert Landt Weihenstephan, September 2005

September 2005

RealReal--Time PCR is based on FluorescenceTime PCR is based on Fluorescence

λ wavelenght (nm)ε extinction coefficientq quantum yield

Absorbtion MaximumEmission MaximumStoke's ShiftQuenchFRET (Fluorescence Resonance Energy Transfer)

PhotobleachingBackground fluorescence Lifetime

Fluorescence is less sensitive when compared to radioactivity or enzyme-linked reactions

September 2005

Real-Time-PCR InstrumentsRealReal--TimeTime--PCR InstrumentsPCR Instruments

7900HTApplied Biosystems

SmartCyclerCepheid

SDS7000Applied Biosystems

RotorGeneCorbett Research

iCyclerBioRad

OpticonMJ Research

SDS7700Applied Biosystems

SuperconvectorAlphaHelixMx3000/4000

Stratagene

iCyclerBioRad

LightTyperRoche Diagnostics

LightCyclerRoche Diagnostics

LightCycler480Roche Diagnostics

MiniOpticonMJ Research-BioRad

September 2005

As a producer of fluorescent probes we are As a producer of fluorescent probes we are interested in new PCR detection technologiesinterested in new PCR detection technologies

September 2005

Principle: adjacent hybridisation and FRET

Fluorescein (donor) LC RED640/705 (acceptor) Phosphate

EXCITATIONNON DETECTED

EMISSIONEXCITATION FRET

DETECTEDEMISSION

Annealing 1-5nt

AMPLICON

Molecular Concepts: Hybridization ProbesMolecular Concepts: Hybridization Probes

September 2005

The probes monitor the actual amount of PCR-product

The slope of the log C(of the target concentration)is proportional to the cycle number.The amount of a unknown sample can easily read out from this standard curve.

How quantification worksHow quantification works

September 2005

RealReal--Time qPCR itself is not very exciting Time qPCR itself is not very exciting

0

200

400

600

800

1000

1200

1400

1994 1995 1996 1997 1998 1999 2000 2001 2002

real-time PCR

Real-Time PCR publications

September 2005

• Single Nucleotide Polymorphism (SNP)• each mismatch destabilizes hybridisation strenght• the melting temperature is lowered for mismatched probes• Use a pair of long (high Tm) anchor and short sensor probe

50 60 70 80 90

50 60 70 80 90 Melting temperature

-dF/

dT

AMPLICON

AMPLICON -dF/

dT

How genotyping worksHow genotyping works

September 2005

Example : hemachromatosis (HFE)Example : hemachromatosis (HFE)Example : hemachromatosis (HFE)

Homozygousmatch

Homozygousmismatch

Heterozygous

September 2005

Example : Identification of bacteriaExample :Example : Identification of bacteriaIdentification of bacteria

Brucella Gram negativeGram positive

September 2005

q(Genoq(Geno--)Typing)TypingDetection of

sequence variations (mutations) and how to find minimal amountsof variants, e.g. contaminations,

minimal residual diseases, or growing resistant populations,

varying in just one base.

September 2005

G A1 : 1G A

G

G

G GG

5 : 1A

G

GG

GG

GG

G

G GG

G GGG

GG

GG

GG

G

GG

GG

GGG

100 : 1A

G G

G

G

Problem : Low-abundance (geno)typesProblem : LowProblem : Low--abundance (geno)typesabundance (geno)types

September 2005

• Several mutations• Ratio: 1:10,000

Melting on artificial targets

Example I : k-ras Codon Gly12Example I : kExample I : k--ras Codon Gly12ras Codon Gly12

Cys

Gly (wt)Asp

September 2005

PNA/LNA-mediated PCR-clampingPNA/LNAPNA/LNA--mediated PCRmediated PCR--clampingclamping

WT 10,000

MU 1

WT 10,000

MU 1

O

O

P

Base

O

O-O

-O O

O

O

P

Base

O

n

HN

N

HN

N

O

O

Base

O

Base

O

n

DNA PNA10,000

PNA

WT

MU 1

September 2005

Molecular concepts: Clamped-Probe-AssayMolecular concepts: ClampedMolecular concepts: Clamped--ProbeProbe--AssayAssay

Possible applications :

• detection of minor variants (e.g. k-ras codon 12,13)• minimal residual diseases (MRD)• developing resistances (STI-571 in abl exon 6 in CML, developing lamivudine resistance in HBV, ...)

50 60 70 80 90

50 60 70 80 90 Melting temperature

-dF/

dT

AMPLICON -dF/

dT

AMPLICON

wt-pecific PNA no signal

September 2005

Example I : k-ras Codon 12Example I : kExample I : k--ras Codon 12ras Codon 12

September 2005

Melting curves from different patientsMelting curves from different patientsMelting curves from different patients

mutant 12Cys

wild type (clamped)

mutant

different clinical samplescontaining k-ras

Codon 12mutations

68°C 65°C61°C

Melting curve (fluorescence vs. temperature)

Melting curve (dFl/dT vs. temperature)

September 2005

Dilution row of mutant in wt DNADilution row of mutant in wt DNADilution row of mutant in wt DNA

1:100,000wt controlw/o PNA

Melting curve (fluorescence vs. temperature)

Melting curve (dFl/dT vs. temperature)

Amplification plot (normalized)

1:10,000

Rapid detection of K-ras mutations in bile by peptide nucleic acid-mediated PCR clamping and melting curve analysis: comparison with restriction fragment length polymorphism analysis. Chen CY, Shiesh SC, Wu SJ. Clin Chem. 2004 Mar;50(3):481-9. Epub 2004 Jan 12

Transrenal DNA as a diagnostic tool: important technical notes. Su YH, Wang M, Block TM, Landt O, Botezatu I, Serdyuk O, Lichtenstein A, Melkonyan H, Tomei LD, Umansky S. Ann N Y Acad Sci. 2004 Jun;1022:81-9

September 2005

Clamping probes consisting of Locked-Nucleic Acid (LNA) work even well

Clamping probes consisting of Clamping probes consisting of LockedLocked--Nucleic Acid (LNA) work even wellNucleic Acid (LNA) work even well

A 3'-terminal attached MB dye boosts the surpression significantly (working concentration is 20-fold lower).

September 2005

Example II : JAK2 mRNA or gen. V617FExample II : JAK2 mRNA or gen. V617FExample II : JAK2 mRNA or gen. V617F

Val+LNA

ValPhe

Phe+LNA

September 2005

Preexistence and evolution of imatinib mesylate-resistant clones in chronic myelogenous leukemia detected by a PNA-based PCR clamping technique. Kreuzer KA, Le Coutre P, Landt O, Na IK, Schwarz M, Schultheis K, Hochhaus A, Dörken B. Ann Hematol. 2003 Apr 12

Example III : Leukemia (CML) STI resistanceExample III : Leukemia (CML) STI resistanceExample III : Leukemia (CML) STI resistance

Clinical resistance to STI-571 cancer therapy caused by bcr-abl gene mutation or amplification. Gorre, M.E., Mohammed, M., Ellwood, K., Hsu, N., Paquette, R., Rao, P.N., Sawyers, C,L. Science, 293: 876-80, 2001

5‘- c-abl GeneBank accession number X16416 -3‘

ABLx4 F

ABLx6 R

ABLx56 FM Sensor [T]s Anchor

STI-T315

Sensor Val

255 Anchor

Y253E255

Sensor 255

The M315T mutation in ABL exon 6 and mutations at Y253 and E255 in ABL exon 4 are responsible for the resistance of bcrABL-clones in CML.

September 2005

Example III : Leukemia (CML) STI resistanceExample III : Leukemia (CML) STI resistanceExample III : Leukemia (CML) STI resistance

Dilution series (bcr/abl Tyr253His)

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

55 60 65 70 75 80 85

temperature [°C]

fluor

esce

nce

1:11:501:1001:500NTCWTCPatient 4

0,000,020,040,060,080,100,120,140,160,18

45 50 55 60 65 70 75 80

Week 0Week 15Week 16Week 21Week 25Week 30WTCNTC

1E-5

1E-4

1E-3

1E-2

1E-1

0 4 8 12 16 20 24 28 32

Patient 1 (Thr315Ile)

HR

Serial dilutions of mutant in wildtype, wildtype control and patient sample. Patient history monitored with the CPA assay.

wt mt

September 2005

Example IV : Growing resistances in infectious diseases - Mycobacteria katG (Isoniazid)

Example IV : Growing resistances in infectious Example IV : Growing resistances in infectious diseases diseases -- Mycobacteria katG (Isoniazid)Mycobacteria katG (Isoniazid)

September 2005

Example IV: Mycobacteria katG –DesignExample IV: Mycobacteria katG Example IV: Mycobacteria katG ––DesignDesign

September 2005

Amplification – Melting Analysis wt/mtAmplification Amplification –– Melting Analysis wt/mtMelting Analysis wt/mt

wt

mt

September 2005

Amplification – Melting Analysis wt/mtAmplification Amplification –– Melting Analysis wt/mtMelting Analysis wt/mt

wt

mt

wt+LNA

September 2005

Amplification – Melting Analysis wt/mtAmplification Amplification –– Melting Analysis wt/mtMelting Analysis wt/mt

wt

mt

wt+LNA

mt+LNA

September 2005

Amplification – Melting Analysis wt/mtAmplification Amplification –– Melting Analysis wt/mtMelting Analysis wt/mt

wt

mt

1:1001:1000

wt

1:1000

wt

mt

September 2005

Expl. V: Plasmodium chloroquine resistanceExpl. V: Plasmodium chloroquine resistanceExpl. V: Plasmodium chloroquine resistance

Use of a locked-nucleic-acid oligomer in the clamped-probe assay for detection of a minority Pfcrt K76T mutant population of Plasmodium falciparum. Senescau A, Berry A, Benoit-Vical F, Landt O, Fabre R, Lelievre J, Cassaing S, Magnaval JF. J Clin Microbiol. 2005 Jul;43(7):3304-8.

September 2005

Example VI – current projects – HPA-1Example VI Example VI –– current projects current projects –– HPAHPA--11

Should all pregnant women be tested for their platelet PLA (Zw, HPA-1) phenotype? Br J Haematol. 1994 Jan;86(1):1-5.

About 97% of the population has the HPA-1a allele

Human platelet antigens (HPA) can be targets for antibody responses that cause life-threatening thrombocytopenia following platelet transfusions or pregnancy.

Search for fetal HPA-1b alleles in the maternal blood(HPA-1a background)

September 2005