WO2011104695A2 - Détection de la mutation de kras dans l'exon 2 par rcp quantitative en temps réel spécifique aux allèles - Google Patents

Détection de la mutation de kras dans l'exon 2 par rcp quantitative en temps réel spécifique aux allèles Download PDF

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Publication number
WO2011104695A2
WO2011104695A2 PCT/IB2011/050819 IB2011050819W WO2011104695A2 WO 2011104695 A2 WO2011104695 A2 WO 2011104695A2 IB 2011050819 W IB2011050819 W IB 2011050819W WO 2011104695 A2 WO2011104695 A2 WO 2011104695A2
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allele
dna
primer
wild type
molecular beacon
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PCT/IB2011/050819
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English (en)
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WO2011104695A3 (fr
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Luca Morandi
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GAMMAGENETICS Sàrl
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Publication of WO2011104695A2 publication Critical patent/WO2011104695A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism

Definitions

  • CRC colorectal cancer
  • mCRC metastatic CRC
  • EGFR epidermal growth factor receptor
  • Cetuximab Erbitux, Merck, Lyon, France
  • cetuximab a monoclonal antibody interfering with the extracellular domain of EGFR
  • tumour regression is more frequently observed among patients with increased EGFR gene copy number (Moroni et al, 2005; Sartore-Bianchi et al, 2007; Cappuzzo et al, 2008), while lack of response and short survival was observed in individuals harbouring a KRAS mutation (LieVre et al, 2006, 2008; Di Fiore et al, 2007; Khambata-Ford et al, 2007; Amado et al, 2008; De Roock et al, 2008).
  • a cancer cell may cany a heterozygous or homozygous KRAS mutation, increasing the genetic heterogeneity of the tissue material used.
  • the present invention is related to a novel method based on allele specific quantitative real time PCR (AS-qPCR) using molecular beacon probes to detect the following mutation of exon 2 of KRAS gene: G12A; G12C; G12D; G12R; G12S; G12V; G113D.
  • AS-qPCR allele specific quantitative real time PCR
  • PRODUCT SIZE 173, PAIR ANY COMPL : 3.00, PAIR 3' COMPL : 2.00
  • Cod12R TGGAGCTCGTGGCGTAGGCA 74 74 66
  • Cod12C AAACTTGTGGTAGTTGGAGCTTGTGGCGTAGGCAA 73 73 66
  • Cod12S AAACTTGTGGTAGTTGGAGCTAGTGGCGTAGGCAA 70 73 66
  • Cod12A AAACTTGTGGTAGTTGGAGCTGCTGGCGTAGGCA 75 74 66
  • Cod12D AAACTTGTGGTAGTTGGAGCTGATGGCGTAGGCAA 73 73 66
  • Cod12V AAACTTGTGGTAGTTGGAGCTGTTGGCGTAGGCAA 73 73 66
  • Cod13D AAACTTGTGGTAGTTGGAGCTGGTGACGTAGGCAA 70 73 66
  • Cod13C AAACTTGTGGTAGTTGGAGCTGGTTGCGTAGGC 70
  • 64 Cod61 Lys GACACAGCAGGTAAAGAGGAGTA 63
  • Cod61 Arg ACACAGCAGGTCGAGAGGAGTA 65 73 68
  • Allele 12R CTTGTGGTAGTTGGAGCT+C 56 67 58
  • Allele 12A TGTGGTAGTTGGAGCTG+C 58 66 56
  • Allele 12D GTGGTAGTTGGAGCTG+A 58 65 56
  • AS-KRASUniR TCATGAAAATGGTCAGAGAAACC 65 68 64
  • Figure 1 represents the nucleic acid folding and hybridization prediction of the PCR target by MFOLD prediction software (www. http://mfold.bioinfo.rpi.edu). Primer regions were not affected by secondary structures and this allowed high PCR efficiency.
  • the fluorescent dyes used in this kit is FAM for BRAF alleles. These fluorophores are compatible with the following Real time thermal cyclers present on the market, but it has been optimized with the Mx3000TM (Stratagene) and with ABI Prism7000TM(Applied Biosystems).
  • Molecular beacons are DNA hybridization probes that form a stem-and-loop structure; the loop portion of the molecule is complementary to the target nucleic acid molecule and the stem is formed by the annealing of complementary arm sequences on the ends of the probe sequence (Tyagi & Kramer 1996) (Fig. 3).
  • a fluorescent marker is attached to the end of one arm and a quencher is attached to the end of the other arm.
  • the quencher is a non-fluorescent chromophore that dissipates the energy that it receives from the fluorophore as heat.
  • free molecular beacons adopt a hairpin structure and the stem keeps the arms in close proximity, resulting in efficient quenching of the fluorophore.
  • molecular beacons When molecular beacons encounter a complementary target at the annealing temperature, they undergo a conformational transition that forces the stem apart and results in the formation of a probe/target hybrid that is longer and more stable than the stem (Bonnet et al. 1999). This separates the fluorophore and the quencher, leading to the restoration of fluorescence, which can be detected, whereas any free molecular beacons remain closed and non-fluorescent. If the target DNA sequence does not exactly match the molecular beacon sequence, hybridization and fluorescence will not occur. This is because the thermodynamic properties of the molecular beacons favour the formation of a hairpin form rather than continued hybridization to a less than perfectly matched target sequence.
  • Molecular beacons require the use of a spectrofluorimetric thermal cycler for real-time detection, but can also be used in an endpoint assay using a fluorimeter. The specificity of this system is such that it can readily detect single nucleotide differences (Giesendorf et al. 1998, Marras et al.1999), something that can be difficult to achieve with the 5'nuclease assay system.
  • Figure 2 The molecular beacons assay. Molecular beacons are designed to be complementary to a sequence in the middle of the expected amplicon. The RT step has been omitted. (A) During the denaturation step, the molecular beacons assume a random coil configuration and fluoresce.
  • tumour samples are non- homogeneous and data from a sample of tumour may not be concordant with other sections from the same tumour. Tumour samples may also contain non-tumour tissue. DNA from non-tumour tissue would not be expected to contain the K-RAS mutations detected by the AlphaReal KRAS Kit.
  • the eluate contains the DNA.
  • PCR product were separated by electrophoresis on 2% agarose gel and purified using the Agencourt CleanSeq magnetic beads kit (Beckman Coulter, Inc., Fullerton, CA). Sequencing analysis was then performed on purified product using the DTCS-Quick start Kit (Beckman Coulter, Inc., Fullerton, CA) and a CEQ2000 XL automatic DNA sequencer. The strands were screened using the forward and reverse primers.
  • AS-qPCR Allele specific quantitative PCR was performed in 25 ⁇ volume containing, 10 pmol (2 ul of the 5 pmol/ul) of each of the forward (depending on the allele, see above for sequence; every primers is 3 'end LNA modified except the wild type allele primer) and universal reverse primers (TCATGAAAATGGTCAGAGAAACC), 5 pmol of beacon probe (1 ul of 5 pmol/ul)( FAM- CCGGTGAAGA+GT+GCCTTGA+CGATA+CAGCACCGG-BH 1 ) , 1 x (12.5 ul) FastStart Universal Probe Master (Rox), additional 2 ul of MgC12 25 mM (final concentration 4 mM), 5 ul of Ultrapure water (Invitrogen), and 2.5 ul of DNA.
  • Cycling conditions consisted of 10 minutes at 95°C to activate the enzyme, 30 seconds at 95°C, 30 seconds at 60°C and 30 seconds at 72°C for a total of 38 cycles in two distinct optical thermal cycler: ABI SDS 7000 and Stratagene Mx3000.
  • Sensitivity of the assay was tested using dilutions of SW620 cell line (ATCC) which is mutated for G12V, spiked in with a pool of healthy female donors DNA (Promega).
  • the following DNA mixtures were tested: 100% dilution: only 5 ng of SW620; 50% dilution: 5 ng of wild type DNA with 5 ng of SW620; 5% dilution: 5 ng of wild type DNA with 250 picogram of SW620; 1% dilution: 5 ng of wild type DNA with 50 picogram of SW620; 0.1% dilution: 5 nanogram of wild type DNA with 5 picogram of SW620. 5 ng of wild type pool DNA.
  • the assay showed high PCR efficiency (slope: - 3.02; R2: 0.9945) and good specificity (12 healthy blood donors were tested finding no false positive results). 90 cases previously tested by direct sequencing were confirmed by AS-qPCR.
  • Whole system failure rate during evaluation tests, 24 samples with 3x the positive cut-off concentration (15 picograms of SW620 cells spiked in with 5 ng of wild type DNA) were evaluated for failure rate.
  • the starting material was formalin fixed paraffin embedded (FFPE) tissue sections of 10 microns from a conventional slide.
  • FFPE formalin fixed paraffin embedded
  • the tumor area was 0.5-1.0 cm wide.
  • the amount of tumoral cells exceeded 50% of total cell population.
  • tumour samples are non-homogeneous and data from a sample of tumour may not be concordant with other sections from the same tumour.
  • Tumour samples may also contain non- tumour tissue. DNA from non-tumour tissue would not be expected to contain the K-RAS mutations detected by the AlphaReal KRAS Kit.
  • the detection limit is conditioned by fixation duration in formalin. If the tissue is fixed more than 24 hours, nucleic acids will be of poor quality. Wild type amplicon should be always positive and it may be used as an internal control of each specimen. Specimens with prolonged fixation time (more than 3 days) failed to give any result. Robustness
  • the raw data of Figure 3 shows the detection limit obtained by spike in wild type DNA a serial dilution of SW620 DNA from 100% to 0.1%.
  • AS-qPCR is based on 3' -LNA modified primers to improve specificity of the assay avoiding aspecific PCR amplification due to mismatch jumping of Taq DNA polymerase, as shown in Figure 4.
  • the newly synthesized amplicon is detected by molecular beacon probe.
  • SW620 sequence of KRAS exon 2 this cell line shows homozygote mutation for G12V (see figure 5).
  • the time required to extract, purify, and quantitate DNA from a specimen may add a day or more to the turnaround times of any genomic DNA analytical assay, the ability of AlphaRealKRAS to identify a KRAS mutation with a complete assay time of 3 hours, compares extremely favorably to the gold standard assay using conventional block thermal cycler PCR, followed by electrophoresis, amplicon purification, PCR sequencing, and high-resolution gel or capillary electrophoresis (typically about 48 hours).
  • High sensitivity (0.1% of mutated alleles in a background of 5 ng of wild type DNA) and high specificity (12 healthy donor DNA were found to be wild type) give to this assay very good performances.
  • the only limit of this assay should be represented by tissue over-fixation by formalin which degrade DNA and it produces adduct.

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  • Chemical & Material Sciences (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

L'invention concerne un procédé servant à conduire une réaction en chaîne de la polymérase spécifique aux allèles qui consiste à soumettre un premier ou un second allèle ou les deux à une réaction en chaîne de la polymérase utilisant une paire d'amorces dans laquelle l'une des amorces est complémentaire dudit premier allèle mais laquelle amorce forme un mésappariement avec ledit second allèle au nucléotide terminal 3' de l'amorce, et utilisant une polymérase de l'ADN, ledit premier allèle étant spécifiquement amplifié mais aucune amplification dudit second allèle ne se produisant.
PCT/IB2011/050819 2010-02-26 2011-02-25 Détection de la mutation de kras dans l'exon 2 par rcp quantitative en temps réel spécifique aux allèles WO2011104695A2 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102367478A (zh) * 2011-10-25 2012-03-07 浙江大学 用于KRAS基因突变分型的ARMS-qPCR检测试剂盒及检测方法
CN102796816A (zh) * 2012-07-06 2012-11-28 陕西北美基因股份有限公司 一种体外准确检测kras基因突变的方法
WO2014052613A2 (fr) * 2012-09-26 2014-04-03 Insight Genetics, Inc. Procédés et compositions associées au séquençage de nouvelle génération et utilisables dans le cadre d'analyses génétiques portant sur les cancers liés à alk
EP2780474A4 (fr) * 2011-11-17 2015-06-17 Rheonix Inc Système et procédés pour analyse moléculaire sélective
CN104805207A (zh) * 2015-04-29 2015-07-29 苏州工业园区为真生物医药科技有限公司 检测kras基因突变的试剂盒及其检测方法
JP2017163889A (ja) * 2016-03-15 2017-09-21 アークレイ株式会社 Ｋ−ｒａｓ遺伝子増幅用フォワードプライマーセット、Ｋ−ｒａｓ遺伝子増幅用キット、Ｋ−ｒａｓ遺伝子増幅方法、多型解析方法、及び薬効判定方法

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JP5095888B2 (ja) * 1999-03-18 2012-12-12 エクシコン エ／エス 特異的ｌｎａプライマによる遺伝子内における突然変異の検出
EP1247815A3 (fr) * 2001-03-25 2003-01-29 Exiqon A/S Oligonucléotides modifiés et leurs utilisations
AU2003229549A1 (en) * 2002-02-26 2003-09-09 Roche Diagnostics Gmbh Improved method for allele-specific pcr
EP2069497B1 (fr) * 2006-11-02 2013-08-14 University of Utah Research Foundation Oligonucleotides a utiliser en pcr specifique d'un allele

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102367478A (zh) * 2011-10-25 2012-03-07 浙江大学 用于KRAS基因突变分型的ARMS-qPCR检测试剂盒及检测方法
CN102367478B (zh) * 2011-10-25 2013-07-31 浙江大学 用于KRAS基因突变分型的ARMS-qPCR检测试剂盒及检测方法
EP2780474A4 (fr) * 2011-11-17 2015-06-17 Rheonix Inc Système et procédés pour analyse moléculaire sélective
CN102796816A (zh) * 2012-07-06 2012-11-28 陕西北美基因股份有限公司 一种体外准确检测kras基因突变的方法
WO2014052613A2 (fr) * 2012-09-26 2014-04-03 Insight Genetics, Inc. Procédés et compositions associées au séquençage de nouvelle génération et utilisables dans le cadre d'analyses génétiques portant sur les cancers liés à alk
WO2014052613A3 (fr) * 2012-09-26 2014-06-12 Insight Genetics, Inc. Procédés et compositions associées au séquençage de nouvelle génération et utilisables dans le cadre d'analyses génétiques portant sur les cancers liés à alk
CN104805207A (zh) * 2015-04-29 2015-07-29 苏州工业园区为真生物医药科技有限公司 检测kras基因突变的试剂盒及其检测方法
JP2017163889A (ja) * 2016-03-15 2017-09-21 アークレイ株式会社 Ｋ−ｒａｓ遺伝子増幅用フォワードプライマーセット、Ｋ−ｒａｓ遺伝子増幅用キット、Ｋ−ｒａｓ遺伝子増幅方法、多型解析方法、及び薬効判定方法

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