PNA BRAF Mutation Detection Kit

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1 - PNA BRAF Mutation Detection Kit Catalog Number KA tests/kit Version: 01 Intended for research use only

2 Introduction and Background Intended use The PNA BRAF Mutation Detection Kit detects the V600E somatic mutation in Valine 600 position of human in BRAF oncogene (Table 1). The PNA BRAF 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 colorectal tissue, thyroid cancer biopsies, and thyroid fine-needle aspiration biopsy (FNAB). Table 1. BRAF mutations detected by the kit No. Reagent Mutation Base change Cosmic No. 1 BRAF PNA mix #3 Val600Glu 1799T>A 476 Cosmic Nos are taken from the Catalogue of Somatic Mutations in Cancer. (http://www.sanger.ac.uk/genetics/cgp/cosmic) Background information The B-type Raf Kinase (BRAF) protein is a serine/threonine kinase that has important roles in cellular proliferation, differentiation, and programmed cell death. A BRAF mutation is commonly found in many human cancers including melanoma, colorectal cancer, lung cancer, and papillary thyroid carcinoma. V600E mutation (thymidine to adenosine change in position 1799 in DNA level) is the most prominent mutation, occurring in 90% of these cancers. Therefore, BRAF V600E mutation may serve as a biomarker for diagnosis, prognosis, and treatment options for cancer patients. Principles and overview The PNA BRAF 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-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. 1

3 Figure 1. Principle of the PNA BRAF Mutation Detection Kit The PNA BRAF Mutation Detection Kit can rapidly detect BRAF 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 1%. 2

4 Material and Method List of components No. Name of component Description Volume 1 Non PNA mix #1 Primers only 250 ul 2 BRAF PNA mix #2 V600 PNA and primers 250 ul 3 BRAF PNA 2X premix PCR reaction premix 1250 ul 4 Clamping control Wild-type DNA 400 ul *Each kit contains enough material to test 50 DNA samples for all mutations. Storage instruction The kit should be kept frozen on arrival and store at -20 C. 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 SYBRGreen dye Table 2. List of real time PCR machines that have been tested Company Model Bio-Rad *CFX 96 Roche Light cycler 480 II ABI ABI 7500 ABI ABI 7900 * Current protocol has been optimized with Bio-Rad CFX 96. For other instruments, minor optimization might be necessary. Warnings and precautions All experiments should be performed under proper sterile condition with aseptic techniques. 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 steps should be performed on ice bath. The KRAS PNA 2X premix solution contains SYBRGreen dye and should be always stored in the dark. If DNA has been extracted from a paraffin block, additional purification steps may be required. PCR tubes should be centrifuged briefly before use. Use extreme caution to prevent contamination of PCR reactions with synthetic control material. 3

5 Procedures Figure 2. Flow of the PNA BRAF 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 PNA BRAF Mutation Detection Kit are paraffin embedded tissues, biopsy tissues or thyroid fine-needle aspiration biopsy (FNAB). 2) We strongly recommend using High Pure PCR Template Preparation Kit (Roche Diagnostics, catalog number ) 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 4ºC for up to 24 hours, or at -20ºC for long term storage. 2. Preparation of the Real-Time PCR Mixture Table 3. Set up reaction mix per one reaction Components Volume (ul) BRAF PNA 2X Premix (#3) 10 Each PNA mix (#1~#2) 3 Extracted DNA (10~25 ng total) or Clamping Control (#4) 7 Total volume 20 1) Spin down the PNA mixes and the 2X Premix solution to ensure that the reagents are at the bottom of the tubes. 2) Prepare 2 PCR tubes for one set of DNA samples to be tested. Label them as S1 and S2. Prepare another set of 2 tubes for Clamping Control (wild-type DNA) and label them as C1 and C2. 3) Add 10 µl of BRAF PNA 2X Premix (#3 from the kit) to each tube. 4) For each PCR tube, add 3 µl of corresponding PNA mix from #1~2 from the kit. For example, S1 and C1 tubes will have #1 Non PNA mix #1, S2 and C2 tubes will have #2 BRAF PNA mix #2. 5) For S1~S2 PCR tubes, add 7 µl of prepared DNA sample (10~25 ng total) to each tube to yield 20 µl final volume. 6) For C1~C2 PCR tubes, add 7 µl of Clamping Control (#4 from the kit). 7) Mix the contents and centrifuge it briefly to spin down to be ready for PCR. 4

6 8) 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 µl to each PCR tube. 3. Real-Time PCR reaction 1) Perform real-time PCR using the cycling conditions described below: One Cycle Pre-denaturation 94 C 5 mins Four-Step Cycle (40 cycles) Denaturation 94 C 30 sec PNA clamping 70 C 20 sec Annealing 63 C 30 sec Extension* 72 C 30 sec 2) Set up the detection for reading SYBRGreen at 72 C. 4. Assessment 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~C2) 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 Non PNA mix #1 (C1) 30 BRAF PNA mix #2 (C2) >33 B. DNA samples 1) Determine Ct values of each sample (S1 and S2). 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 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. iii. If the Ct value of Non PNA mix (S1) is greater than 35, 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 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 (30 Ct <35), 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. 5

7 Table 5. The acceptability of the test Acceptability Ct value of S1 Descriptions and recommendations Optimal 22< Ct <30 The amplification and the amount of DNA sample are optimal. Acceptable 30 Ct <35 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 Possibility of false positive is high. Repeat the PCR reaction with a lower amount of DNA. 35 Ct The amplification was failed. Check DNA amount and purity. New DNA prep might be required. 2) Calculate the Ct-1 values by subtracting the sample Ct values from the Standard Ct values. If the Ct of DNA samples is displayed as NA (not applicable), then set Ct value as 38 for further calculation. * Ct-1 = [Standard Ct] [Sample Ct (S2)] where Standard Ct = 35 3) Calculate Ct-2 [Ct value of sample subtracted by Ct value of Non PNA mix]. ** Ct-2 = [Sample Ct (S2)] - [Non PNA mix Ct (S1)] 4) Assess the result along with the values of Ct-1 and Ct-2 as given in Table 6. Table 6. Assessment of the result Ct-1 Ct-2 Assessment 2 Ct-1 All value BRAF V600E mutant 0 Ct-1<2 Ct-2 3 BRAF V600E mutant 3< Ct-2 Wild-type Ct-1<0 All value Wild-type 6

8 Examples of analysis 1) Profile of Clamping Control and DNA sample ; Sample Non PNA mix #1 ; Sample BRAF PNA mix #2 ; Clamping control Non PNA mix #1 ; Clamping control BRAF PNA mix # Table 7. Example of sample Ct values Sample No. Sample 1 Sample 2 Standard * Ct-1 ** Ct-2 Assay (Ct) (Ct) Ct Non PNA mix #1 (S1) BRAF PNA mix #2 (S2) (5) - - * Ct-1 = [Standard Ct] [Sample Ct] ** Ct-2 = [Sample Ct] - [Non PNA mix Ct (S1)] 2 Table 8. Analysis of data Sample No. Sample 1 Sample 2 Assay Ct-1 Ct-2 Ct-1 Ct-2 BRAF PNA mix #2 (S2) Results BRAF V600E mutant Wild-type 1. When Ct-1 is equal to or greater than 2, the sample is assessed to be mutated ( ). 2. If Ct-1 is equal to or greater than 0 and less than 2 ( ), then Ct-2 needs to be determined. 3. If Ct-2 is less than 3, the sample is assessed to be mutated ( ). 5 7

9 References 1. National Cancer Center, Korea Annual Report of the Korea Central Cancer Registry National Health Insurance Corporation Annual Report of Disease Registry Ezzat et al., Thyroid incidentalomas. Prevalence by palpation and ultrasonography. Arch Intern Med. 1994;154: Meier et al., Thyroid nodules: pathogenesis, diagnosis, and treatment. Baillieres Best Pract Res Clin Endocrinol Metab. 2000;14: Kang et al., Prevalence, clinical and ultrasonographic characteristics of thyroid incidentalomas. Thyroid. 2004;14: Rapp et al., Structure and biological activity of v-raf, a unique oncogene transduced by a retrovirus. Proc Natl Acad Sci USA 1983:80:

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