EGFR mutations were defined as exon 19 deletion or exon 21 point mutations. We excluded patients with other more uncommon EGFR mutation profiles. Genotyping of EGFR and KRAS was performed by peptide nucleic acid (PNA)-mediated PCR clamping methods, such as the PNAClampTM EGFR MutationDetection Kit and PNAClampTM KRAS Mutation Detection Kit (PANAGENE, Inc., Daejeon, Korea), using real-time PCR [24 (link)]. PCR was performed in a total reaction volume of 20 μl including the template DNA, primer and PNA probe sets, and SYBR Green PCR master mix. The PCR control lacked the PNA probe and contained the wild-type template. The CFX96 PCR detection system was used to perform PCR.
Pnaclamp kras mutation detection kit
Manufactured by Panagene
Sourced in Cameroon
The PNAClamp KRAS mutation detection kit is a laboratory equipment product designed for the detection of KRAS gene mutations. It utilizes a Peptide Nucleic Acid (PNA) clamping technology to selectively amplify wild-type KRAS sequences, allowing for the identification of specific KRAS mutations.
Automatically generated - may contain errors
Lab products found in correlation
Pnaclamp egfr mutation detection kit, by Panagene (2 mentions)
Cfx96 pcr detection system, by Bio-Rad (2 mentions)
Qiaamp dna ffpe tissue kit, by Qiagen (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
8 protocols using pnaclamp kras mutation detection kit
1
Detecting EGFR and KRAS Mutations
2
Evaluating KRAS Mutation Detection
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To evaluate the feasibility of KRAS molecular testing in biobanked specimens, KRAS exon 2 (codon12 and 13) mutation analysis by PNA-mediated real-time PCR clamping was performed on biobanked frozen tissue in all specimens. The amount of DNA used for PNA clamping was 40-80 ng/test (10 ng/reaction). A PNAClamp TM KRAS Mutation Detection Kit (Panagene, Daejeon, Korea) was used to detect KRAS mutations by real-time PCR, according to the manufacturer's instruction. Briefly, all reactions included template DNA, a primer and PNA probe set, and the SYBR Green PCR master mix. Real-time PCR was performed using a CFX96 PCR detection system (Bio-Rad, Philadelphia, PA, USA). The efficiency of PCR clamping was determined by measuring the threshold cycle (Ct) values. Higher ΔCt (ΔCt = Ct standard -Ct specimen ) values meant that the mutant DNA was efficiently amplified. A cutoff value of 2.0 was used to indicate the presence of mutant DNA. KRAS mutation status of all 45 biobanked frozen tissues that was performed at this time was compared with the results of corresponding FFPE tissues that was initially performed at the time of pathologic diagnosis.
3
KRAS Mutation Detection in CRC Tissues
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DNA was extracted from tumor tissues obtained from 65 patients with CRC, using the QIAamp® DNA FFPE Tissue Kit (Qiagen, Hilden, Germany). KRAS mutations in the CRC tissue samples were detected using a peptide nucleic acid (PNA) PCR mixture (20 µL) containing 7 µL of extracted DNA, 3 µL of PNA mixture, and 10 µL of KRAS PNA premixture, which were provided in the PNAClamp™ KRAS Mutation Detection Kit (Panagene, Daejeon, South Korea). Real-time PCR was performed using the StepOnePlus Real-Time PCR System (Applied Biosystems). The PCR conditions were as follows: 94 °C for 5 min, followed by 40 cycles of 94 °C for 30 s, 70 °C for 20 s, 63 °C for 30 s, and 72 °C for 30 s.
4
Molecular Profiling of Cancer Genes
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EGFR gene alteration was detected by either real-time PCR with PNA-clamping methods, direct sequencing, or both methods. The PNA-Clamp™EGFR Mutation Detection kit (PANAGENE, Inc., Daejeon, Korea) was used for real-time PCR, performed as described[45 (link)]. When detection was done only with direct sequencing, exon 18, 19, 20, and 21 were sequenced as previously described[44 (link)]. When both methods were used, exons containing mutations detected by real-time PCR were sequenced, and exon 19 was sequenced if no mutation was detected by real-time PCR.
KRAS gene alteration was also detected by either real-time PCR with PNA-clamping methods, direct sequencing, or both methods. The PNA-Clamp™KRAS Mutation Detection kit (PANAGENE, Inc., Daejeon, Korea) was used for real-time PCR, performed as described[46 ]. KRAS exon 2, which contains codons 12 and 13, was sequenced by direct sequencing as previously described[44 (link)].
ALK gene alteration was detected by immunohistochemistry or fluorescence in situ hybridization(FISH)[47 (link)].
KRAS gene alteration was also detected by either real-time PCR with PNA-clamping methods, direct sequencing, or both methods. The PNA-Clamp™KRAS Mutation Detection kit (PANAGENE, Inc., Daejeon, Korea) was used for real-time PCR, performed as described[46 ]. KRAS exon 2, which contains codons 12 and 13, was sequenced by direct sequencing as previously described[44 (link)].
ALK gene alteration was detected by immunohistochemistry or fluorescence in situ hybridization(FISH)[47 (link)].
5
Real-time PCR-based KRAS Mutation Detection
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The PNA Clamp KRAS Mutation Detection kit (PANAGENE, Daejeon, Korea) was used to detect KRAS mutations in codon 12 and 13 by real-time PCR according to the manufacturer's instructions, as previously described.28 (link) Finally, ΔCt values were calculated as follows: ΔCt1 = [standard Ct]−[sample Ct], and ΔCt2 = [sample Ct]−[non-PNA mix Ct]. A higher ΔCt value meant that the mutant was efficiently amplified. A cut-off value of 2.0 was used to determine the presence of mutant DNA.
6
PNA Clamp KRAS Mutation Detection
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A PNA Clamp KRAS Mutation Detection Kit (Panagene Inc., Daejeon, Korea) was used to detect KRAS mutations in codons 12 and 13 with real-time polymerase chain reaction (PCR) according to the manufacturer’s instructions. Real-time PCR reactions were performed on a CFX96 PCR detection system (Bio-rad, Philadelphia, PA, USA). The efficiency of PCR clamping was determined by measuring the threshold cycle (Ct) values. Mutation status was determined based on a Ct value difference < 2 between the control and sample.
7
KRAS Mutation Analysis in Colorectal Cancer
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The study was based on tumor tissue from 14 FFPE CRCs (from the archive of the Department of Patholo-gy of Pomeranian Medical University) in which KRAS mutations had been previously detected with the PNAClamp KRAS mutation detection kit (Panagene, Daejeon, Korea) using manual microdissection. Details of clinicopathological characteristics of the study group are presented in Table I. Altogether there were one G1, 11 G2 and two G3 adenocarcinomas.
8
KRAS Mutation Detection in FFPE Samples
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In total the DNA from 28 samples (14 from the luminal and 14 from the peripheral areas) was isolated with the QIAamp DNA FFPE tissue kit (Qiagen, Hilden, Germany) and the amount of DNA was measured with a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific Wilmington, USA). Afterwards the presence of KRAS mutations was detected with the PNAClamp KRAS mutation detection kit (Panagene, Daejeon, Korea). Accuracy of results of KRAS testing in the laboratory was validated by successfully passing the ESP KRAS EQA scheme.
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