Probe Based Detection of Genetic Variations - Screening and in-vitro Diagnostics qpcr 2009 Freising, Olfert Landt, Berlin, Germany

Transcription

1 Probe Based Detection of enetic Variations - Screening and in-vitro Diagnostics qpcr 2009 Freising, Olfert Landt, Berlin, ermany

2 Real-Time PCR worldwide well established? Dear sir, i am Bader. I worked in molecular and infectious desi departement in central lab and blood bank in Riyadh for 2 years.but now im working in histopathology in central lab and blood bank. We would like to open molecular in our section.for tumor markers.olso CMV,HPV,TB etc.on these days we are prepair the place.and we will get light cycler (ROCHE) insturment soon. If you do not mind? please send the all tumor markers you have,and we will bay the kits later. PCR was invented my first cycler was 1988 the Pharmacia ATAQ and I know Real-Time PCR since 1996 (Perkin Elmer LS50B). Now after 13+ years we should expect that this is not longer new and we might wonder about the number of registrations for qpcr meetings.

3 Real-Time PCR worldwide well established? Dear Sascha, Thank you for the information. By "how the antisense primer works" I wonder what is the mechanism between the antisense primer, the DNA polymerase and the template? From what I can see from the article, the two primers make one PCR product. Is it correct that one DNA strand is transcribed? I hope this formulation is improved. Best regards, PhD student from Norway PCR was invented join the Nobel Price Laureate lecture 16:30 today!

4 Some Important Remarks (1) Real-Time PCR is not better in sensitivity (2) Real-Time PCR Makes agarose gel analysis needless (at least in routine) = convenience factor (3) Real-Time PCR gives quantity information (4) Real-Time PCR allows to multiplex tests (multi color reading) Most important (Real-Time) PCR applications are : Detection (quantification) and Typing (identification)

5 Quantification and Typing Application Detection Quantification Identification Typing (Variations) Target (examples) Pathogens, Fusion Transcripts (usually unique sequence in a different background) as above mrna (splicing variants can be similar to the background) any (through probe binding) SNP, Deletions/Insertions Splice Variants Pathogens (eventually mixed polulations) IVD Requirements Detection Limit (LOD) Linear Range Reporting Range Accuracy Detection Limit (LOD)

6 Typing Etablished PCR Methods Typing Methods Specific Primer + Probe TaqMan probe Molecular Beacons FRET Hybridization Probes SimpleProbe, LightUp HyNA probes Scorpion Primers High Resolution Melting HRM + unlabelled probe Working Principle Variant-specific amplification and detection of the amplicon Digestion based on binding Competitive binding Competitive binding or Melting Curve Analysis Melting Curve Analysis Looped binding of the amplicon Melting Curve Analysis Melting Curve Analysis Pro Sensitive (minor variants) Mainstream on demand Precise (not common) Differentiated Results Sensitive (minor variants) Low costs Differentiated Results

7 Hybridization Probes - df/dt Target The ct-values are proportional to the amount of target Target - df/dt Melting temperature HSV-1/2 Quantification Typing (Probe Melting)

8 Typing Applications (limitations) Typing Methods Limitations Pro Specific Primer + Probe TaqMan probe Molecular Beacons Hybridization Probes SimpleProbe et cetera Scorpion Primers HRM HRM + unlabelled probe Read-through events False positive results False results for new SNP One probe per SNP needed Not working for minor variants Not working for minor variants One primer per SNP needed False results for new SNP Undifferentiated results Limited multiplexing Not working for minor variants Sensitive (minor variants) Mainstream on demand Precise (not common) Differentiated Results Sensitive (minor variants) Low costs Differentiated Results

9 Multiple SNP : Lactose Intolerance

10 Multiple SNP : Lactose Intolerance hetero C-13913T hetero C-13910T wt mt C High-Resolution melting gives unclear results which are not correclty recognized and assorted by the instrument software.

11 Multiple SNP : Lactose Intolerance LightCycler hybridization probes yield specific and reproducible melting temperatures for different variants. TaqMan probes will fail and report a false genotype.

12 Unexpected variations : MTHFR % Sensitivity + Specificity HRM analysis (published ESH 2008)

13 Unexpected variations : MTHFR x heterozygotes! heterozygote C677T heterozygote C677T High-Resolution melting gives unclear results which are not correclty recognized and assorted by the instrument software.

14 Unexpected variations : MTHFR 677 heterozygote C677T T (mt) heterozygote C677T High-Resolution melting gives unclear results which are not correclty recognized and assorted by the instrument software.

15 Unexpected variations : MTHFR 677 heterozygote C677T heterozygote C677T C (wt) High-Resolution melting gives unclear results which are not correclty recognized and assorted by the instrument software.

16 Unexpected variations : MTHFR 677 heterozygote C677T heterozygote C677T C (wt) A685 High-Resolution melting gives unclear results which are not correclty recognized and assorted by the instrument software.

17 Unexpected variations : MTHFR 677 Two patient samples analyzed 2005 and 2008 in Pennsylvania showed a new intermediate melting point; sequencing revealed the up to now not known variant A685 (not published).

18 Complex enotyping (4 variants per channel) Codon 141 (Nor98) 610 Left : Codon 136 (3 alleles)+141, Right: 171 (4 alleles) + 175E LightMix 480HT Scrapie Susceptibility Mutation Detection Kit 12 allele variants in 5 codons, 384 ovine samples within 80 min, up to 2,500 animals per day

19 Real-Time PCR applications are limited to 5 (6 claimed) results (dye channels) or 6-12 results (3 dyes melting points)

20 Typing Applications (limitations) Typing Methods Limitations Pro Specific Primer + Probe TaqMan probe Molecular Beacons Hybridization Probes SimpleProbe et cetera Scorpion Primers HRM HRM + unlabelled probe Read-through events False positive results False results for new SNP One probe per SNP needed Not working for minor variants Not working for minor variants Limited multiplexing > 3-4 types per channel One primer per SNP needed Undifferentiated results Limited multiplexing Sensitive (minor variants) Mainstream on demand Precise (not common) Differentiated Results Sensitive (minor variants) Low costs Differentiated Results

21 Combination with Array Based Typing Workflow Real-Time PCR (45-60 min) Hybridization and Detection (45 min) Examples Pathogens : Dengue Virus, Adenovirus, HPV Resistance : Mycobacteria kat + rpob Mutations Clamping Assay Colon Cancer : k-ras Codon 12/13 Mutations

22 ... Workflow

23 Dengue TaqMan Assay & enotyping 1-41

24 Adenovirus LightCycler Serogroups A-FA

25 Papilloma Virus Typing (32 HPV Types) Combination with SYBR reen or TaqMan LightCyler PCR Two primer mixes generating amplicons of ~430 or ~125 bp for formalin-fixed, paraffin-embedded (FFPE) tissue sections 45 min Protocol

26 Mycobacteria kat and rpob genes

27 Typing Applications (limitations) Typing Methods Limitations Pro Specific Primer + Probe TaqMan probe Molecular Beacons Hybridization Probes SimpleProbe et cetera Scorpion Primers HRM HRM + unlabelled probe Read-through events False positive results False results for new SNP One probe per SNP needed Not working for minor variants Not working for minor variants One primer per SNP needed False results for new SNP Undifferentiated results Limited multiplexing Not working for minor variants Sensitive (minor variants) Mainstream on demand Precise (not common) Differentiated Results Sensitive (minor variants) Low costs Differentiated Results

28 Problem : Low-abundance (geno)types A A 1 : 1 A 5 : 1 A 100 : 1

29 Detection of Mutations in HIV-1

30 Sensitive detection of k-ras k mutations from colorectal cancer (CRC) specimen In 2007 Amgen launched the drug Vectibix, an anti-efr antibody for the therapy of CRC Patients with mutations in the k-ras gene do not respond to therapy. The EMEA authority made patient k-ras mutation tests mandatory. Problem : Detection of minimal amounts of sequence mutations varying in only one base.

31 Molecular Concepts: Clamped-Probe-Assay wt-pecific competitor AMPLICON - df/dt no signal AMPLICON - df/dt Melting temperature 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 Jun;1022:81-9 Detection of Point Mutations in Kirsten ras 2 ene Using Locked Nucleic Acids Clamped PCR Beranek, Bures, Sacha, Sakra, Rajman, Jandik, Rudolf, Landt Chem. Listy 101, (2007)

32 Melting curves for different mutations

33 LCD Array k-ras k Codon 12/13 Nr. Specificity Nr. Specificity Nr. Specificity Nr. Specificity Nr. Specificity 1 WT-5 4 Cod-12-Val 7 Cod-12-Ser 10 Cod-13-Asp C Hyb-Control 2 WT-Center 5 Cod-12-Ala 8 Cod-12-Cys 11 Cod-13-Arg 3 WT-3 6 Cod-12-Asp 9 Cod-12-Arg 12 Cod-13-Cys

34 Example II : BRAF V617F

35 Example III : CML livec Resistance Melting Curve Analysis exhibits a signal for week 16 fluorescence 0,8 0,7 0,6 0,5 0,4 0,3 0,2 Dilution series (bcr/abl Tyr253His) wt mt 1:1 1:50 1:100 1:500 NTC WTC Patient 4 0,18 0,16 0,14 0,12 0,10 0,08 0,06 0,04 0,02 0,00 1E-1 1E-2 Week 0 Week 15 Week 16 Week 21 Week 25 Week 30 WTC NTC Patient 1 (Thr315Ile) 0, E-3 1E-4 HR temperature [ C] Dilution row to determine the limit of detection, Patient Sample 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 E Quantitative PCR reveals an increase after week 28

36 Example IV : Tamiflu resistance

37 Summary There are many different Real-Time PCR typing methods established. TaqMan probe and Scorpion primer based assays might miss unexpected variants and need to include one probe (channel) per variant. HRM is cost effective but the results are undifferentiated and of limited use in diagnostics (IVD). LightCycler SimpleProbe and hybridization probes give a differentiated picture of highly variable gene regions. Hybridization probes allow a moderate multiplex testing due to combination of reading melting point temperatures and different dye channels Hybridization probes can be combined with competitor probes to detect minor variants in cancer and resistant polulations of pathogens. Real-Time PCR methods can be easily combined with low-density arrays to achieve a positive/negative result or at least a semi-quantitative result with a highly differentiated information about the genotype, variant or mutation.

38 Fight Cancer Olfert Landt - Berlin