PNAClamp Mutation Detection Kit For in vitro diagnostic use Instruction manual for product # PNAC-3001 Version 1.6 Store at -15 C to -20 C Instruction Version: Ver. 1.6 Date of Revision: 2012 Dec 1 / 19 PNG-PCEUM001
Contents Intended Use... 3 Background Information... 3 Principles and Overview... 5 Contact Information... 6 Additional Equipment and Reagents Required... 6 Warnings and Precautions... 7 Components of the PNAClamp Mutation Detection Kit... 8 Procedures... 9 1. DNA Preparation... 9 2. Preparation of the Real-Time PCR Mixture... 10 3. Real-Time PCR reaction... 10 4. Assessment... 11 Examples of Analysis... 13 References... 17 Explanation of Symbols... 18 Endnotes... 19 2 / 19 PNG-PCEUM001
PRODUCT NAME Product Name: PNAClamp Mutation Detection Kit Brand Name: PNAClamp Mutation Detection Kit PNAClamp Mutation Detection Kit PNAClamp Mutation Detection Kit Please read the instructions carefully prior to use. The PNAClamp Mutation Detection Kit is a CE marked diagnostic device in accordance with the European Union in vitro Diagnostic Medical Device Directive 98/79/EC. It is KFDA approved for clinical use in Korea. INTENDED USE The PNAClamp Mutation Detection Kit is to detect 29 somatic mutations in the epidermal growth factor receptor () gene (Table 1). The 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 biopsies and surgical tissue samples. The kit is for in vitro diagnostic use. BACKGROUND INFORMATION The epidermal growth factor receptor () is a family member of receptor tyrosine kinases, expressed on the surface of epidermal cells. Overexpression or overactivation of is linked to a number of cancers, including lung cancer, anal cancers and glioblastoma multiform. The PNAClamp Mutation Detection Kit detects most prevalent mutations described to date in the gene, including T790M, the presence of which correlates with resistance to tyrosine kinase inhibitors. Detecting somatic mutations in gene may provide a useful strategy to predict the response to the tyrosine kinase inhibitors in efforts to increase the survival rate of lung cancer patients receiving targeted therapy. 3 / 19 PNG-PCEUM001
Table 1. mutations detected by the kit No. Reagent Exon Amino Acid Change Nucleotide change Cosmic No. 1 G719X PNA mix 2 E19 del. PNA mix del: deletion mutation, ins: insertion mutation 18 p.g719a c.2156g>c 6239 18 p.g719s c.2155g>a 6252 18 p.g719c c.2155g>t 6253 19 p.e746_a750del c.2235_2249 del 15 6223 19 p.e746_t751>i c.2235_2252 > AAT 13551 19 p.e746_t751del c.2236_2253 del 18 12728 19 p.e746_t751>a c.2237_2251 del 15 12678 19 p.e746_s752>a c.2237_2254 del 18 12367 19 p.e746_s752>v c.2237_2255>t 12384 19 p.e746_a750del c.2236_2250 del 15 6225 19 p.e746_s752>d c.2238_2255 del 18 6220 19 p.l747_a750>p c.2238_2248 >GC 12422 19 p.l747_t751>q c.2238_2252 >GCA 12419 19 p.l747_e749del c.2239_2247 del 9 6218 19 p.l747_t751del c.2239_2253 del 15 6254 19 p.l747_s752del c.2239_2256 del 18 6255 19 p.l747_ A750>P c.2239_2248 TTAAGAGAAG>C 12382 19 p.l747_p753>q c.2239_2258 >CA 12387 19 p.l747_t751>s c.2240_2251 del 12 6210 19 p.l747_p753>s c.2240_2257 del 18 12370 19 p.l747_t751del c.2240_2254 del 15 12369 19 p.l747_t751>p c.2239_2251>c 12383 3 T790M PNA mix 20 p.t790m c.2369c>t 6240 4 S768I PNA mix 20 p.s768i c.2303g>t 6241 20 p.v769_d770insasv c.2307_2308 ins9 12376 5 E20 Ins.3dup PNA mix 20 p.h773_v774insh c.2319_2320 inscac 12377 6 E20 Ins. 3 PNA mix 20 p.d770_n771insg c.2310_2311 insggt 12378 7 L858R or L861Q PNA mix 21 p.l858r c.2573t>g 6224 21 p.l861q c.2582t>a 6213 Cosmic Nos are taken from the Catalogue of Somatic Mutations in Cancer. (http://www.sanger.ac.uk/genetics/cgp/cosmic) 4 / 19 PNG-PCEUM001
PRINCIPLES AND OVERVIEW The PNAClamp 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. 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-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. Figure 1. Principle of the PNAClamp Mutation Detection Kit The kit can rapidly detect mutations (within 2 h) with high sensitivity even with a small amount of DNA (25~50 ng). The detection limit of the kit, when the mutated gene is mixed with wildtype DNA, is less than 1%. 5 / 19 PNG-PCEUM001
CONTACT INFORMATION The PNAClamp Mutation Detection Kit should be kept frozen on arrival. For any questions including technical support or concerns, please contact the distributors or the manufacturer. Manufacturer Manufacturer: PANAGENE Inc. 54, Techno 10-ro, Yuseong-gu, Daejeon, 305-510, Korea Email: info@panagene.com EC Representative: PentaGen, s.r.o Ke Klinku 143, 27351 Horni Bezdekov, Czech Republic Email: info@pentagen.cz ADDITIONAL EQUIPMENT AND REAGENTS REQUIRED Reagents and equipment for DNA extraction 0.2 ml DNase-free PCR tubes or plates Pipettes A real-time PCR instrument fitted with a detector enabling evaluation of SYBR Green dye Table 2. List of compatible real time PCR instruments Company Model Bio-Rad CFX 96 Roche Light cycler 480 II ABI ABI 7500 ABI ABI 7900 Qiagen Rotor-Gene Q For other instruments, minor optimization might be necessary. 6 / 19 PNG-PCEUM001
WARNINGS AND PRECAUTIONS Please read the instruction carefully and become familiar with all components of the kit prior to use. All experiments should be performed under proper sterile conditions with aseptic techniques. It recommended that users have separate, dedicated pipettes and filter pipette tips to add DNA template and during the preparation of reagents. Always wear powder-free gloves when you handle the kit. 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. All experimental procedures should be performed at room temperature. However, exposing PNA 2X premix at room temperature should be minimized for the optimal amplification. Dissolve reagents completely and mix them thoroughly by vortex. The 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 weakly centrifuged before use. Using non-recommended volume for reagents not only result in loss of performance but also increase the chance of false result. 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. Do not exchange and mix up different lots or other manufacture s product. Upon using instruments, use only recommended consumables only. If not, instruments will not be usable or false result may prominent. Additional validation testing by user may necessary when using non-recommended instruments. Do not re-use any remaining reagents after PCR amplification is completed. Do not use the reagents beyond the expiry date. 7 / 19 PNG-PCEUM001
Components of the PNAClamp Mutation Detection Kit Store at -15 C to -20 C No. Name of component Description Volume Cap label 1 Non PNA mix Primers only 100 µl 2 G719X PNA mix G719X PNA and primers 100 µl 3 E19 del. PNA mix E19 del. PNA and primers 100 µl 4 T790M PNA mix T790M PNA and primers 100 µl 5 S768I PNA mix S768I PNA and primers 100 µl 6 E20 Ins. 3 dup PNA mix E20 Ins. 3 dup PNA and primers 100 µl 7 E20 Ins. 3 PNA mix E20 Ins. 3 PNA and primers 100 µl 8 L858R or L861Q PNA mix L858R and L861Q PNA and primers 9 PNA 2X premix PCR reaction premix 100 µl 1,250 µl /vial, 2 vials 10 Clamping control Wild-type DNA 600 µl 1 2 3 4 5 6 7 8 2X premix Control *Each kit contains enough material to test 25 DNA samples for all mutations. 8 / 19 PNG-PCEUM001
PROCEDURES Figure 2. Workflow of the PNAClamp 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) Paraffin embedded tissues or biopsy tissues can be used as specimens. 2) Specimen transport: Use standard pathology methodology to ensure specimen quality. 3) For DNA extraction, High Pure PCR Template Preparation Kit (Roche Diagnostics, catalog number 11796828001) is recommended. 4) Extracted DNA can be stored at 4ºC for up to 24 hours, or at -20 ºC for long term storage. 9 / 19 PNG-PCEUM001
2. Preparation of the Real-Time PCR Mixture Table 3. Set up reaction mix per one reaction Components Volume PNA 2X Premix (#9) 10 µl Each PNA mix (#1~#8) 3 µl Extracted DNA (25~50 ng total) or Clamping control (#10) 7 µl Total volume 20 µl 1) Prepare 8 PCR tubes for one set of DNA samples to be tested. Label them as S1, S2, S3, S4, S5, S6, S7 and S8. Prepare another set of 8 tubes for Clamping control (wild-type DNA) and label them as C1~C8. 2) Add 10 µl of PNA 2X Premix (#9 from the kit) to each tube. 3) For each PCR tube, add 3 µl of corresponding PNA mix from #1~8 from the kit. For example, S1 and C1 tubes will have #1 Non PNA mix, S2 and C2 tubes will have #2 G719X PNA mix and so forth. 4) For S1~S8 PCR tubes, add 7 µl of prepared DNA sample (25~50 ng total) to each tube to yield 20 l final volume. 5) For C1~C8 PCR tubes, add 7 µl of Clamping control (#10 from the kit). 6) If you have more than one DNA sample 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 µl 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. 3. Real-Time PCR reaction 1) Perform real-time PCR using the cycling conditions described below: ONE CYCLE Pre-denaturation 94 ºC 5 min FOUR-STEP CYCLING (40 CYCLES) Denaturation 94 ºC 30 sec PNA clamping 70 ºC 20 sec Annealing 63 ºC 30 sec Extension* 72 ºC 30 sec * Set up the detection for reading SYBR Green at 72ºC. 10 / 19 PNG-PCEUM001
4. Assessment * Refer to the specialized instrument user guide by Panagene 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 Clamping control (tube C1~C8) 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 Assay Acceptable Ct range 1 Non PNA mix (C1) 30 2 G719X PNA mix (C2) > 32 3 E19 del. PNA mix (C3) > 32 4 T790M PNA mix (C4) > 31 5 S768I PNA mix (C5) > 28 6 E20 Ins. 3 dup PNA mix (C6) > 28 7 E20 Ins. 3 PNA mix (C7) > 28 8 L858R or L861Q PNA mix (C8) > 31 B. DNA samples 1) Determine Ct values of each sample (S1~ S8). i. Ct value of Non PNA mix (S1) should be 22~35. ii. Ct value of Non PNA mix (S1) can serve as an internal control to indicate the purity and the concentration of DNA. Thus, the validity of the test can be decided by the Ct value of Non PNA mix (S1) as shown in Table 5. Table 5. The acceptability of samples Acceptability Ct value of Non PNA mix(s1) Optimal 22< Ct <30 Acceptable 30 Ct <35 Descriptions and recommendations The amplification and the amount of DNA sample are optimal. The target gene was amplified with low efficiency. For more reliable result, it is suggested that repeat PCR reaction with a higher amount of DNA. Invalid Ct 22 35 Ct Possibility of false positive is high. Repeat the PCR reaction with a lower amount of DNA. The amplification was failed. Check DNA amount and purity. New DNA prep might be required. 11 / 19 PNG-PCEUM001
2) Calculate the ΔCt-1 values by subtracting sample Ct values from standard Ct values given in Table 6 below. If the Ct of samples is displayed as NA (not applicable), then set Ct value as 38 for further calculation. *ΔCt-1 = [Standard Ct] [Sample Ct (S2, S3, S4, S5, S6, S7 or S8)] Table 6. Standard Ct values for mutation detection Assay Standard Ct 2 G719X PNA mix (S2) 34 34 3 E19 del. PNA mix (S3) 34.5** 4 T790M PNA mix (S4) 33 5 S768I PNA mix (S5) 30 6 E20 Ins. 3 dup PNA mix (S6) 30 7 E20 Ins. 3 PNA mix (S7) 30 8 L858R or L861Q PNA mix (S8) 33 **If you use Light Cycler 480 II or ABI7900, please use the standard Ct value 34.5 for 3 E19 del. PNA mix (S3) instead of 34. 3) Calculate ΔCt-2 [Ct value of sample subtracted by Ct value of Non PNA mix]. **ΔCt-2 = [Sample Ct (S2, S3, S4, S5, S6, S7 or S8)] - [Non PNA mix Ct (S1)] 4) Assess the result along with the values of ΔCt-1 and ΔCt-2 as given in Table 7. Table 7. Assessment of the result ΔCt-1 ΔCt-2 Assessment 2 ΔCt-1 All value Mutant 0< ΔCt-1 <2 ΔCt-2 3 Mutant 3< ΔCt-2 Wild ΔCt-1 0 All value Wild 12 / 19 PNG-PCEUM001
EXAMPLES OF ANALYSIS 1. Using Bio-Rad CFX 96 1) Profile of Clamping Control Assay Clamping control Ct Accep. range Result 1 Non PNA (C1) 23.31 30 Acceptable 2 G719X (C2) 36.28 > 32 Acceptable 3 E19 del. (C3) 34.93 > 32 Acceptable 4 T790M (C4) 36.43 > 31 Acceptable 5 S768I (C5) 32.91 > 28 Acceptable 6 E20 Ins. 3 dup (C6) 30.23 > 28 Acceptable 7 E20 Ins. 3 (C7) 36.37 > 28 Acceptable 8 L858R or L861Q (C8) 33.49 > 31 Acceptable 2) Profile of samples 1 ; Non PNA mix 2 ; G719X PNA mix 3 ; E19 del. PNA mix 4 ; T790M PNA mix 5 ; S768I PNA mix 6 ; E20 Ins. 3 dup PNA mix 7 ; E20 Ins. 3 PNA mix 8 ; L858R or L861Q PNA mix 13 / 19 PNG-PCEUM001
Table 8. Example of sample Ct values Assay Sample No. Sample 1 Ct Sample 2 Ct Sample 3 Ct 1 Non PNA (S1) 24.45 22.74 22.35 Standard Ct **ΔCt-2 *ΔCt-1 2 G719X (S2) 33.61 35.64 34.40 34 (9) 2-1 9-2 3 E19 del. (S3) 29.09 34.26 24.02 34 (10) 3-1 10-3 4 T790M (S4) 33.36 27.35 33.22 33 (11) 4-1 11-4 5 S768I (S5) 31.24 32.17 32.56 30 (12) 5-1 12-5 6 E20 Ins. 3 dup (S6) 32.63 30.36 28.36 30 (13) 6-1 13-6 7 E20 Ins. 3 (S7) 35.28 35.29 35.73 30 (14) 7-1 14-7 8 L858R or L861Q (S8) 32.58 27.19 31.98 33 (15) 8-1 15-8 *ΔCt-1 = [Standard Ct] [Sample Ct] **ΔCt-2 = [Sample Ct] [Non PNA mix Ct (S1)] Table 9. Analysis of data Assay Sample No. Sample 1 Sample 2 Sample 3 ΔCt-2 ΔCt-1 ΔCt-2 ΔCt-1 ΔCt-2 ΔCt-1 2 G719X (S2) 9.16 0.39 12.9-1.64 12.05-0.4 3 E19 del. (S3) 4.64 4.91 11.52-0.26 1.67 9.98 4 T790M (S4) 8.91-0.36 4.61 5.65 10.87-0.22 5 S768I (S5) 6.79-1.24 9.43-2.17 10.21-2.56 6 E20 Ins. 3 dup (S6) 8.18-2.63 7.62-0.36 6.01 1.64 7 E20 Ins. 3 (S7) 10.83-5.28 12.55-5.29 13.38-5.73 8 L858R or L861Q (S8) 8.13 0.42 4.45 5.81 9.63 1.02 Result Exon 19 deletion T790M & L858R or L861Q Exon 19 deletion 1. When ΔCt-1 is equal to or greater than 2, the sample is assessed to be mutated ( ). 2. If ΔCt-1 is greater than 0 and less than 2 ( ) and ΔCt-2 is equal to or less than 3, the sample is assessed to be mutated ( ). 14 / 19 PNG-PCEUM001
2. Using ABI7900 1) Profile of Clamping Control Assay Clamping control Ct Accep. range Result 1 Non PNA (C1) 24.98 30 Acceptable 2 G719X (C2) 36.42 > 32 Acceptable 3 E19 del. (C3) 36.81 > 32 Acceptable 4 T790M (C4) 36.75 > 31 Acceptable 5 S768I (C5) 32.31 > 28 Acceptable 6 E20 Ins. 3 dup (C6) 30.80 > 28 Acceptable 7 E20 Ins. 3 (C7) 35.08 > 28 Acceptable 8 L858R or L861Q (C8) 36.72 > 31 Acceptable 2) Profile of samples 1 ; Non PNA mix 2 ; G719X PNA mix 3 ; E19 del. PNA mix 4 ; T790M PNA mix 5 ; S768I PNA mix 6 ; E20 Ins. 3 dup PNA mix 7 ; E20 Ins. 3 PNA mix 8 ; L858R or L861Q PNA mix 15 / 19 PNG-PCEUM001
Table 10. Example of sample Ct values Assay Sample No. Sample 1 Ct Sample 2 Ct Sample 3 Ct 1 Non PNA (S1) 27.78 27.22 26.54 Standard Ct **ΔCt-2 *ΔCt-1 2 G719X (S2) 34.19 33.68 33.52 34 (9) 2-1 9-2 3 E19 del. (S3) 38.00 28.86 36.10 34.5 (10) 3-1 10-3 4 T790M (S4) 35.40 33.55 35.23 33 (11) 4-1 11-4 5 S768I (S5) 32.49 31.91 33.39 30 (12) 5-1 12-5 6 E20 Ins. 3 dup (S6) 35.49 34.83 35.40 30 (13) 6-1 13-6 7 E20 Ins. 3 (S7) 37.48 32.77 35.69 30 (14) 7-1 14-7 8 L858R or L861Q (S8) 34.45 33.55 26.64 33 (15) 8-1 15-8 *ΔCt-1 = [Standard Ct] [Sample Ct] **ΔCt-2 = [Sample Ct] [Non PNA mix Ct (S1)] Table 11. Analysis of data Sample No. Sample 1 Sample 2 Sample 3 Assay ΔCt-2 ΔCt-1 ΔCt-2 ΔCt-1 ΔCt-2 ΔCt-1 2 G719X (S2) 6.41-0.19 6.46 0.32 6.98 0.48 3 E19 del. (S3) 10.22-3.50 1.64 5.64 9.56-1.60 4 T790M (S4) 7.62-2.40 6.33-0.55 8.69-2.23 5 S768I (S5) 4.71-2.49 4.69-1.91 6.85-3.39 6 E20 Ins. 3 dup (S6) 7.71-5.49 7.61-4.83 8.86-5.40 7 E20 Ins. 3 (S7) 9.70-7.48 5.55-2.77 9.15-5.69 8 L858R or L861Q (S8) 6.67-1.45 6.33-0.55 0.10 6.36 Result Wild Exon 19 deletion L858R or L861Q 1. When ΔCt-1 is equal to or greater than 2, the sample is assessed to be mutated ( ). 2. If ΔCt-1 is greater than 0 and less than 2 ( ) and ΔCt-2 is equal to or less than 3, the sample is assessed to be mutated ( ). 16 / 19 PNG-PCEUM001
REFERENCES 1. Kim et al., Detection and comparison of peptide nucleic acid-mediated real-time polymerase chain reaction clamping and direct gene sequencing for epidermal growth factor receptor mutations in patients with non-small cell lung cancer. Lung Cancer. 75 (3):321-325, 2012. 2. Han et al., Detection of Mutation Status in Lung Adenocarcinoma Specimens with Different Proportions of Tumor Cells Using Two Methods of Differential Sensitivity. J Thorac Oncol. 7 (2):355-64, 2012. 3. Choi et al., PNA-mediated Real-Time PCR Clamping for Detection of Mutations. Bull. Korean Chem. Soc. 31 (12): 3525-3529, 2010. 4. Lee et al., PNA-Mediated PCR Clamping for the Detection of Mutations in Non- Small Cell Lung Cancer. Tuberc Respir Dis. 69:271-278, 2010 5. Noro et al., Gefitinib (IRESSA) sensitive lung cancer cell lines show phosphorylation of Akt without ligand stimulation. BMC Cancer 6: 277, 2006. 17 / 19 PNG-PCEUM001
EXPLANATION OF SYMBOLS Batch Code Use by Manufacturer EC Representative In Vitro Diagnostic Medical Device Catalogue number Temperature Limitation PANAGENE Inc. 54, Techno 10-ro, Yuseong-gu, Daejeon, 305-510, Korea PentaGen, s.r.o Ke Klinku 143, 27351 Horni Bezdekov, Czech Republic 18 / 19 PNG-PCEUM001
ENDNOTES Information in this document is 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. 54, Techno 10-ro, Yuseong-gu, Daejeon, 305-510, Korea Tel: +82-42-861-9296 www.panagene.com 19 / 19 PNG-PCEUM001