PNAClamp™ KRAS Mutation Detection Kit (Ver.2)
Contents
1. PANAGENE PNAClamp KRAS Mutation Detection Kit PNAClamp KRAS Mutation Detection Kit Ver 2 For research use only Instruction manual for product PNAC 1002 Version 4 0 Store at 15 C to 20 C Instruction Version Ver 4 0 Date of Revision 2012 May 14722 PNG PCKRMO002 PANAGENE PNAClamp KRAS Mutation Detection Kit Table of Contents Ii rz C M BL c 3 BACK Or OU Talon Ati OM asas E 4 Principles and OV CLF VIC W i vccecetisccecssnsccsssissisassinvecseethsececesssccessvssieassbeeeeeee0sstensssusdsvesussieeseueeeceuelesteassesscerss 5 Contact LAMON eege 6 Additional Equipment and Reagents Required sscccscscccssssssssssssssssssssssssscsscsccccccccsscssssssssssees 6 Warnmes and WT ivsssss e I 7 Components of the PNAClamp KRAS Mutation Detection Kit 0oo000000000000000000000000000000000ene 7 POC COUN CS sisa anaa na aan Waaa a 8 DNA PG ar O aaa a aa a ET UU aaa a aaa a 8 2 Preparation of the Real Time PCR Mixture ooo000000000000000000000000000000000000000000000000000000000 000000000 9 RM CSI indu egi iuit M 10 s MR E mee 10 Examples or AAAI SIS sisian aana a ag UNG Na tin Ever rave T cas Ir EE ANNA TAN ue QU NEGEN AN PEE E2. B DNA samples 1 Determine Ct values of each sample S1 53 i Ct value of Non PNA mix S1 should be 22 34 ii Ct value of Non PNA mix S1 can serve as an internal to indicate the purity and the concentration of sample DNA Thus the validity of the test can be decided by the Ct value of Non PNA mix S1 as shown in Table 5 10 22 PNG PCKRMO002 PANAGENE PNAClamp KRAS Mutation Detection Kit in If the Ct value of Non PNA mix S1 is equal to or greater than 34 target gene was not successfully amplified and therefore the result is not reliable Check DNA amount and purity New DNA prep might be required iv If the Ct value of Non PNA mix S1 is equal to or less than 22 the result may be false positive Repeat the PCR reaction with lower amount of DNA v If the Ct value of Non PNA mix S1 is between 30 and 35 30x Ct lt 34 the target gene was amplified with low efficiency For more reliable result it is suggested to repeat PCR reaction with a higher amount of DNA Table 5 The acceptability of the test Acceptability Ct value of S1 Descriptions and recommendations The ae lification and the amount of DNA sample are The target gene was amplified with low efficiency For more Acceptable 30 lt Ct lt 34 reliable result it is suggested that repeat PCR reaction with a higher amount of DNA sch Let The amplification was failed Check DNA amount and purity en New DNA prep might be required Ct 22 Poss
3. z E I ls ed n 9 E amp EE ILS m s OSS 6 ui fo SS E B eeben 29 Di Lt a x e Sample 3 HB gu 3 ion E z oon pe a o O Sample Non PNA mix 1 2 Sample KRAS PNA mix 2 3 Sample KRAS PNA mix 3 4 Clamping control Non PNA mix 1 5 Clamping control KRAS PNA mix 2 Clamping control KRAS PNA mix 3 Table 12 Example of sample Ct values Non PNA mix 1 S1 24 72 25 11 24 74 pf KRAS PNA mix 2 S2 28 34 35 00 35 00 35 D 3 KRAS PNA mix 3 S3 38 00 32 75 35 00 35 8 ACt Standard Ct Sample Ct ACt 2 Sample Ct Non PNA mix Ct S1 15722 PNG PCKRMO002 PANAGENE PNAClamp KRAS Mutation Detection Kit Table 13 Analysis of data Sample No Sample 1 Sample 2 Sample 3 KRAS PNA mix 2 S2 10 26 CARS KRASPNA 13 28 3 00 764 WH 10 26 mix 3 S3 KRAS codon 12 KRAS codon 13 Wild type mutant type mutant type When ACt 1 is equal to or greater than 2 the sample is assessed to be mutated E If ACt 1 is greater than O and less than 2 4 then ACt 2 needs to be determined If ACt 2 is less than 6 the sample is assessed to be mutated HM O29 e Final assessments are as follows Table 14 Final assessment of the result of sample DNA Sample No KRAS PNA mix42 KRAS PNA mix 3 mutant type mutant type 16 22 PNG PCKRMO002 me S PANAGENE PNAClamp KR
4. Comparison of peptide nucleic acid mediated PCR and direct sequencing in formalin fixed paraffin embedded tissue Pathol Res Pract 207 12 762 8 2011 Yoon et al K ras Gene Mutation in Non Small Cell Lung Cancer J Lung Cancer 1 1 55 59 2002 Beau Faller et al Detection of K Ras mutations in tumour samples of patients with non small cell lung cancer using PNA mediated PCR clamping British Journal of Cancer 100 6 985 992 2009 Chang et al Fast simultaneous detection of K RAS mutations in colorectal cancer BMC cancer 9 179 2009 Dabritz et al Detection of Ki ras mutations in tissue and plasma samples of patients with pancreatic cancer using PNA mediated PCR clamping and hybridisation probes British Journal of Cancer 92 2 405 412 2005 Behn et al Facilitated detection of oncogene mutations from exfoliated tissue material by a PNA mediated enriched PCR protocol J Pathol 190 1 69 75 2000 Hilger et al The Ras Raf MEK ERK Pathway in the Treatment of Cancer Onkologie 25 6 511 518 2002 Bachireddy et al Getting at MYC through RAS Clin Cancer Res 11 12 4278 4281 2005 21 22 PNG PCKRMO002 PANAGENE PNAClamp KRAS Mutation Detection Kit ENDNOTES This product is for research only not for a diagnostic tool or for any other use The kit itself or any of the components in the kit cannot be modified resold or transferred without the approval of manufacturer Information in this document i
5. DNA duplexes even with a single mismatch PNA will not bind to complementary DNA strand unlike DNA Second PNA oligomers are not recognized by DNA polymerases and will not be utilized as primers in subsequence real time PCR Instead it serves as a sequence selective clamp that prevents amplification during subsequent PCR When there is a mutation in target gene and therefore a mismatch is present the DNA PNA duplex is destabilized allowing strand elongation from a bound DNA oligomer which serves as a PCR primer The outcome is the positive reaction in real time PCR from the samples harboring mutant allele while amplification of the wild type gene is suppressed L C gt D i Perfectly matched PNA e e X e of probe for wild type E Real time PCR He Amplicen Perfectly matched PNA be for wild type tg e Figure 1 Principle of the PNAClamp KRAS Mutation Detection Kit Real time PCR 53 22 PNG PCKRM002 PANAGENE PNAClamp KRAS Mutation Detection Kit The PNAClamp M KRAS Mutation Detection Kit can rapidly detect KRAS mutation within 2 h with high sensitivity even with a small amount of DNA 10 25 ng The detection limit of the kit when the mutated gene is mixed with wild type background is less than 146 CONTACT INFORMATION The PNAClamp M KRAS Mutation Detection Kit should be kept frozen on arrival For any questions including technical support or concerns please contact the
6. distributors or the manufacturer Manufacturer Panagene Inc 816 Tamnip dong Yuseong gu Email info panagene com Daejeon 305 510 South Korea Tel 82 42 861 9296 ADDITIONAL EQUIPMENT AND REAGENTS REQUIRED Y Reagents and equipment for DNA extraction Y 0 2 ml DNase free PCR tubes or plates v Pipettes Y Areal time PCR instrument fitted with a detector enabling evaluation of SYBR Green dye Table 2 List of real time PCR machines that have been tested Light cycler 480 I For other instruments minor optimization might be necessary 6 22 PNG PCKRM002 S PANAGENE PNAClamp KRAS Mutation Detection Kit WARNINGS AND PRECAUTIONS v All experiments should be performed under proper sterile conditions with aseptic techniques v Always wear powder free gloves when you handle the kit V To avoid repeated freezing and thawing aliquot all reagents into appropriate volumes and store frozen until use Thaw appropriate volumes of reagents before each experiment S All experimental procedures should be performed at room temperature However exposing KRAS PNA 2X premix at room temperature should be minimized for the optimal amplification Dissolve reagents completely and mix them thoroughly by vortexing The KRAS PNA 2X premix solution contains fluorescence dye and should be kept dark If DNA has been extracted from a paraffin block additional purification steps may be required PCR tubes should be centrifuged briefly before
7. mixes SE e KH Et Analysis 10 min Real Time PCR 2h Figure 2 Flow of the PNAClamp KRAS Mutation Detection Kit 1 DNA preparation Specimen collection and DNA extraction reagents are not included in the kit and should be provided by the user 1 Specimens that can be analyzed using the PNAClamp M KRAS Mutation Detection Kit are paraffin embedded tissues or biopsy tissues 2 We strongly recommend using High Pure PCR Template Preparation Kit Roche Diagnostics catalog number 11796828001 for DNA extraction especially for paraffin embedded tissues 3 Specimen transport Use standard pathology methodology to ensure specimen quality 4 Extracted DNA can be stored at 4 C for up to 24 hours or at 20 C for long term storage 8 22 PNG PCKRMO002 SS u PANAG EN PNAClamp KRAS Mutation Detection Kit 2 Preparation of the Real Time PCR Mixture Table 3 Set up reaction mix per one reaction KRAS PNA 2X Premix 4 10 ul Extracted DNA 10 25 ng total or Clamping Control 5 1 Prepare 3 PCR tubes for one set of DNA samples to be tested Label them as S1 S2 and 53 Prepare another set of 3 tubes for Clamping Control wild type DNA and label them as C1 C3 2 Add 10 ul of KRAS PNA 2X Premix 4 from the kit to each tube 3 For each PCR tube add 3 ul of corresponding PNA mix from 1 3 from the kit For example Sl and Cl tubes will have 1 Non PNA mix 1 S2 and C2 tubes will have 2 KRAS PNA mix 2 and so f
8. use Use extreme caution to prevent contamination of PCR reactions with synthetic control material NN S S S S Using non recommended volume for reagents not only result in loss of performance but also increase the chance of false result RW Using non recommended volume and concentration for target DNA sample not only result in loss of performance but also increase the change of false result Y Avoid mixing with different lots or other manufacture s product v Upon using instruments use only recommended consumables only If not instruments will not be usable or false result may prominent v Additional validation testing by user may necessary when using non recommended instruments S Do not re use any remaining reagents after PCR amplification is completed Y Do not use the reagents beyond the expiry date Components of the PNAClamp KRAS Mutation Detection Kit Store at 15 C to 20 C KRAS PNA mix 2 Codon 12 PNA and primers 120 ul KRAS PNA mix 3 Codon 13 PNA and primers 120 ul KRAS PNA 2X premix PCR reaction premix 1 250 ul Clamping control Wild type DNA 250 ul bach kit contains enough material to test 30 DNA samples for all mutations 1122 PNG PCKRM002 S PANAGENE PNAClamp KRAS Mutation Detection Kit PROCEDURES OHands on Time Winstrument Time Amplification of target DNA by N N Analysis of N Real Time PCR Pp Results Total time about 3 h Preparation Preparation of PCR
9. AS Mutation Detection Kit 3 Using ABI 7900 1 Profile of Clamping Control and DNA sample Aus etm oer cum gut g Arga ain an Sample 1 Sample 2 Sample Non PNA mix 1 Sample KRAS PNA mix 2 Sample KRAS PNA mix 3 Clamping control Non PNA mix 1 Clamping control KRAS PNA mix 2 Clamping control KRAS PNA mix 3 000906961 Table 15 Example of sample Ct values Sg 2 KRAS DNA mix 2 S2 24 95 35 76 36 88 34 D ACt Standard Ct Sample Ct ACt 2 Sample Ct Non PNA mix Ct S1 17722 PNG PCKRM002 PANAGENE PNAClamp KRAS Mutation Detection Kit Table 16 Analysis of data Sample No Sample 1 Sample 2 Sample 3 DKRAS PNA 10 92 11 93 mix 2 S2 KRAS PNA 10 57 11 75 mix 3 S3 KRAS codon 12 KRAS codon 13 Wild type mutant type mutant type When ACt 1 is equal to or greater than 2 the sample is assessed to be mutated LA If ACt 1 is greater than O and less than 2 CM then ACt 2 needs to be determined If ACt 2 is less than 6 the sample is assessed to be mutated g ge oe ocu x Final assessments are as follows Table 17 Final assessment of the result of sample DNA Sample No KRAS PNA mix 2 KRAS PNA mix 3 mutant type mutant type 18 22 PNG PCKRMO002 AN 49 PANAGENE PNAClamp KRAS Mutation Detection Kit 4 Using ABI 7500 1 Profile of Clamping Control and DNA sample Amplification Plot Amplification Plo
10. H NUN EE BUNGA a ANG NA HU PEE EINE IEEE 13 IROL OE CNC CG aaa suna aaa a a aa 21 PV OU CS ee 22 2422 PNG PCKRMO002 PANAGENE PNAClamp KRAS Mutation Detection Kit PNAClamp KRAS Mutation Detection Kit Please read the instructions carefully prior to use INTENDED USE The PNAClamp KRAS Mutation Detection Kit is an in vitro diagnostic test to detect seven somatic mutations in the KRAS oncogene Table 1 The PNAClamp KRAS Mutation Detection Kit is to be used by trained laboratory professionals within a laboratory environment using for example DNA extracted from formalin fixed paraffin embedded samples of lung and colorectal biopsies and surgical tissue samples The kit is for research use only Table 1 KRAS mutations detected by the kit p G12D c 35G A p G12A c 35G C KRAS PNA p G12V c 35G T mix 42 p G12S c 34G gt A p G12R c 34G gt C p G12C c 34G gt T a p G13D c 38G gt A 532 mix 3 Cosmic Nos are taken from the Catalogue of Somatic Mutations in Cancer http www sanger ac uk genetics CGP cosmic 3 22 PNG PCKRMO002 PANAGENE PNAClamp KRAS Mutation Detection Kit BACKGROUND INFORMATION The KRAS mutation is found in several cancers including colorectal lung thyroid and pancreatic cancers and cholangiocarcinoma KRAS mutations are often located within codons 12 and 13 of exon 2 which may lead to abnormal growth signaling by the p21 r
11. NA mix 3 4 Clamping control Non PNA mix 1 5 Clamping control KRAS PNA mix 2 Clamping control KRAS PNA mix 3 Table 9 Example of sample Ct values ee Nol Sample 1 Sample 2 Sample 3 Sample 4 i ad Po x D Non PNA mix 1 S1 28 84 wn 22 27 38 26 18 T oe ACt 1 Standard Ct Sample Ct ACt 2 Sample Ct Non PNA mix Ct S1 13 22 PNG PCKRM002 PANAGENE PNAClamp KRAS Mutation Detection Kit Table 10 Analysis of data Sample No Sample 1 Sample 2 Sample 3 Sample 4 KRAS PNA 224 13 78 mix 2 S2 ERASE 65g 2446 71 10 62 2 mix 3 S3 KRAS codon 12 KRAS codon 12 KRAS codon 13 and x mutant type mutant type TOMOS KRAS codon 13 mutant type When ACt 1 is equal to or greater than 2 the sample is assessed to be mutated 7 If ACt 1 is greater than O and less than 2 L1 then ACt 2 needs to be determined If ACt 2 is less than 6 the sample is assessed to be mutated g oe aps s Final assessments are as follows Table 11 Final assessment of the result of sample DNA Sample No KRAS PNA mix 2 KRAS PNA mix 3 mutant type mutant type KRAS codon 12 and Sample 4 Mutant Mutant KRAS codon 13 mutant type 14 22 PNG PCKRMO002 me S PANAGENE PNAClamp KRAS Mutation Detection Kit 2 Using Roche LC480 1 Profile of Clamping Control and DNA sample 4 Sample 1 B Sample 2 e a Im a m a 19 f IER fim P Pw Gi
12. as protein These alterations in cell growth and division may trigger cancer development as signaling is excessive A KRAS mutation often serves as a useful prognostic marker of drug response For example a KRAS mutation 15 considered to be a strong prognostic marker of response to tyrosine kinase inhibitors such as gefitinib Iressa or erlotinib Tarceva Recently KRAS mutations have been detected in many colorectal cancer patients and may be associated with responses to cetuximab Erbitux or panitumumab Vectibix which are used in colon cancer therapy 4 22 PNG PCKRM002 49 PANAG ENE PNAClamp KRAS Mutation Detection Kit PRINCIPLES AND OVERVIEW The PNAClamp M KRAS Mutation Detection Kit is based on peptide nucleic acid PNA mediated real time PCR clamping technology PNA is a synthetic DNA analog in which the phosphodiester backbone is replaced by a peptide like repeat formed by 2 aminoethyl glycine units Since PNA contains no charged phosphate groups the binding between PNA DNA is stronger than between DNA DNA due to the lack of electrostatic repulsion In addition PNA is resistant to DNases and proteases and is stable at wide range of pH PNA mediated real time PCR clamping relies on the following two unique properties of PNA probes First PNA will hybridize to its complementary DNA target sequence only if the sequence is in complete match Since PNA DNA duplexes are more thermodynamically stable than the corresponding DNA
13. ibility of false positive is high Repeat the PCR reaction i with a lower amount of DNA Invalid 2 Calculate the ACt 1 values by subtracting the sample Ct values from the Standard Ct values given in Table 6 If the Ct of DNA samples is displayed as NA not applicable then set Ct value as 38 for further calculation ACt Standard Ct Sample Ct S2 or S3 Table 6 The value of Standard Ct Standard Ct Instrument KRAS PNA mix 2 KRAS PNA mix 3 3 Calculate ACt 2 Ct value of sample subtracted by Ct value of Non PNA mix ACt 2 Sample Ct S2 or S3 Non PNA mix Ct S1 11 22 PNG PCKRMO002 PANAGENE PNAClamp KRAS Mutation Detection Kit 4 Assess the result for each KRAS PNA mix along with the values of ACt 1 and ACt 2 as given in Table 7 Table 7 Assessment of the result for each KRAS DNA mix S2 or S3 0 ACt 1 lt 2 6s ced acci so Wes 5 Assess the result along with the result for each KRAS PNA mix as given in Table 8 Table 8 Final assessment of the result of sample DNA mutant type mutant type KRAS codon 12 and Mutant Mutant KRAS codon 13 mutant type 12722 PNG PCKRMO002 AN 49 PANAGENE PNAClamp KRAS Mutation Detection Kit EXAMPLES OF ANALYSIS 1 Using Bio Rad CFX96 1 Profile of Clamping Control and DNA sample Amplification Amplification gampel 0 1 0 e Sample 2 Q Sample Non PNA mix 1 2 Sample KRAS PNA mix 2 3 Sample KRAS P
14. orth 4 For 51 53 PCR tubes add 7 ul of prepared DNA sample 10 25 ng total to each tube to yield 20 ul final volume 5 For C1 C3 PCR tubes add 7 ul of Clamping Control 5 from the kit 6 If you have more than one DNA samples to be tested prepare one set of clamping control for the entire experiment In such case it is recommended to prepare a master mix containing 2X Premix and each PNA mix for all the samples and to aliquot 13 ul to each PCR tube 7 When all reagents are loaded tightly close seal the PCR tube or 96 well plate Otherwise any remaining reagents may evaporate 9 22 PNG PCKRM002 PANAGENE PNAClamp KRAS Mutation Detection Kit 3 Real Time PCR reaction Perform real time PCR using the cycling conditions described below ONE CYCLE Pre denaturation 5 min FOUR STEP CYCLING 40 CYCLES 2 Setup the detection for reading SYBR Green at 72 C 4 Assessment Refer to the instrument user manual for detail analysis method A Clamping Control wild type DNA control 1 Determine Ct value from each PCR reaction The cycle number at which a signal is detected above background fluorescence is termed as the cycle threshold Ct 2 The Ct values of the wild type DNA control tube C1 C3 should fall in the range given in Table 4 The assay should be repeated if the values are not in recommended range Table 4 The acceptable Ct ranges of Clamping Control D Non PNA mix 1 C1 234X lt 27
15. s subject to change Panagene assumes no responsibility for any errors that may appear in this document No other warranties of any kind express or implied including without limitation implied warranties of merchantability or fitness for a particular purpose are provided by Panagene Panagene shall have no liability for any direct indirect consequential or incidental damages arising out of the use the results of use or the inability to use this product PANAGENE Inc 816 Tamnip dong Yuseong gu Daejeon 305 510 Korea Tel 82 42 861 9296 WWW panagene com 22 22 PNG PCKRMO002
16. t wo Sample 1 Sample 2 1 000 000 ei E 1 000 000 0 000 Ee mm d UE Amplification Plot 1 600 000 H 1 250 000 E ode 3 Q Sample KRAS PNA mix 1 2 Sample KRAS PNA mix 2 3 Sample KRAS PNA mix 3 4 Clamping control Non PNA mix 1 Clamping control KRAS PNA mix 2 Clamping control KRAS PNA mix 3 Table 18 Example of sample Ct values 2 KRAS PNA mix 2 S2 28 05 37 34 38 60 35 D 3 KRAS PNA mix 3 S3 38 00 32 82 37 63 35 8 ACt 1 Standard Ct Sample Ct ACt 2 Sample Ct Non PNA mix Ct S1 19 22 PNG PCKRM002 PANAGENE PNAClamp KRAS Mutation Detection Kit Table 19 Analysis of data Sample No Sample 1 Sample 2 Sample 3 KRAS PNA mix 2 S2 KRAS PNA 95 3 00 7 49 mix 3 S3 KRAS codon 12 KRAS codon 13 Results mutant type mutant type When ACt 1 is equal to or greater than 2 the sample is assessed to be mutated 1 If ACt 1 is greater than O and less than 2 I3 then ACt 2 needs to be determined If ACt 2 is less than 6 the sample is assessed to be mutated g x oun Final assessments are as follows Table 20 Final assessment of the result of sample DNA Sample No KRAS PNA mix 2 KRAS PNA mix 3 mutant type mutant type 20 22 PNG PCKRMO002 PANAGENE PNAClamp KRAS Mutation Detection Kit REFERENCES l Choi et al Frequency of KRAS BRAF and PIK3CA mutations in advanced colorectal cancers
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