FACTOR V of LEIDEN REAL TIME

Transcription

1 When you have completed this, the graph will show all of the wells identified as shown in the legend. Homozygous WT Negative Heterozygous Homozygous MUT FACTOR V of LEIDEN REAL TIME REF Opening report tab you can read the fluorescence and the genotyping for all sample. production: CLONIT S.r.l. via B. Quaranta 57, Milano - Italy Tel Fax CLONIT srl Via Quaranta 57, MILAN ITALY Tel Fax info@clonit.it website: Printed: 08 th November 2004 ST pag.16/16

2 Introduction 1 Page Factor V of Leiden 3 Aim of the kit 4 Detection of point mutation 4 Allelic discrimination 4 Kit description 5 Sample material 5 Kit storage and stability 5 Kit Performance 5 Kit Contents 6 Materials Required but Not Supplied 6 For all samples, identify the alleles by using the arrow tool or the lasso tool and the selections on the call menu. Highlight all the wells in the matrix at the bottom of the windows. ST pag.2/16 ST pag.15/16

3 In the graph, select the controls by clicking the arrow tool and dragging a rectangle around the population. From the call menu, select the allele. Allele Y: Wild type control Allele X: Mut control Allele X&Y: Etero control Call menù Arrow tools Introduction Factor V of Leiden Before 1993, the evaluation of inherited thrombophilia was limited to functional assays for protein C, protein S, and antithrombin III, but abnormal results from these tests identified only 7% of patients with Venous thrombotic events (VTE) (3). Laboratory evaluation of thrombophilia was revolutionized when Dahlback and colleagues described a new and very common inherited thrombophilic syndrome: hereditary resistance to activated protein C (APC). APC has the ability to cleave and inactivate activated clotting Factors V and VIII. The researchers assessed the disorder by measuring the ratio of activated partial thromboplastin time (aptt) clotting times with and without the addition of APC (4). Soon afterwards, Bertina and colleagues discovered that a single, well-conserved G to A missense mutation at nucleotide 1691 of the Factor V gene was responsible for APC resistance (5). The resulting amino acid substitution-arginine to glutamine at amino acid 506-is located at one of the sites in Factor V that is recognized, cleaved, and inactivated by APC. Commonly known as Factor V Leiden, the so-called R506Q mutation results in a predisposition to thrombosis by creating an inefficiently inactivated the clotting factor. Unlike the genetic heterogeneity in patients with protein C, protein S, or antithrombin III deficiency, greater than 95% of those with functional APC resistance, as measured by the clotting time test, have the identical Factor V R506Q missense mutation. Shortly after its discovery, Factor V Leiden became known as the most common inherited cause of thrombophilia, with one copy of the mutated gene present in approximately 20% of unselected VTE patients, up to half of those with recurrent or familial VTE, and in 3%-7% of the general U.S. population. It is significantly less common in those of non-european ancestry. Heterozygous carriers of Factor V Leiden carry a three- to seven-fold increased risk of thrombosis, while homozygotes carry a 50- to 100-fold increased risk. The very high prevalence of APC resistance suggests that its presence should be evaluated in all patients undergoing thrombophilia workups. The typical APC resistance-screening test consists of a ratio of functional aptt clotting times in the presence and absence of exogenous APC and has a sensitivity and specificity approaching 100%. The confirmatory test for APC resistance, a direct DNA-based assay of the causative point mutation, should be performed on all patients with low or borderline APC resistance assays to distinguish heterozygotes from homozygotes. Typically a PCRbased method is used to detect the Factor V R506Q point mutation in DNA (6). In contrast to the continuous values obtainable with the APC resistance assay, the Factor V Leiden direct mutation test definitively determines the presence of zero, one, or two copies of the disease-causing mutation. In addition to its role in the clinical evaluation of thrombophilia, the direct DNA assay for Factor V Leiden is the preferred testing method for the evaluation of asymptomatic at-risk family members. Although asymptomatic Factor V Leiden-carrying relatives will not be routinely anticoagulated unless they have a history of thrombosis, they should be counseled to consider anticoagulant prophylaxis before specific, predictable periods of increased ST pag.14/16 ST pag.3/16

4 thrombotic risk such as surgery, pregnancy, oral contraceptive use, or prolonged immobilization. Aim of the Kit The Factor V of Leiden Real Time kit permits the individuation of Factor V mutation of Leiden associate to Ereditary Trombophilia. Rapidity of this Method permits the test esecution in a small time. Allelic Discrimination The software graphs the results of the allelic discrimination run on a scatter plot of allele X (Mut) Rn versus allele Y (wild type) Rn. To check results click result tab and select allelic discrimination. Highlight the controls in the wells in the matrix at the bottom of the windows. Detection of Point Mutations Mutation Gene Location Nucleotidic Aminoacidic Substitution Substitution G1691A Factor V Exone 10 G-1691 A Arg Glu Allelic Discrimination The Allelic discrimination exploits the 5 nuclease activity of DNA Taq Polymerase to cleave a MGB probes during PCR. The MGB probes contains a reporter dye at the 5 end of the probe and a no fluorescent quencer (NFQ) group at the 3. Fluorogenic probes with the MGB (minor groove binder) attached to the 3 end perform well in the 5 nuclease assay. They are an improvement over unmodified probes because shorter sequences (13-to 20-mers) can be used to obtain probe that have an optimal Tm (65 to 67). Thus, attachment of the MGB enables the use of shorter fluorogenic probes, which results in improved mismatch discrimination. Mismatches between a probe and target reduce the efficiency of probe hybridizaton. Furhermore, Taq Polymerase is more likely to displace the mismatched probe rather than cleave to it, releasing the reporter dye. During the reaction, cleavage of the MGB probes separates the reporter dye and the NFQ group, which results in increased fluorescence of the reporter. Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye. The allelic discrimination utilize two MGB Probes. Identify the alleles by using the arrow tool or the lasso tool and the selections on the call menu. complementary to the wild type portion complementary to the mutated portion FAM-MGB Probe VIC-MGB Probe Post read ST pag.4/16 ST pag.13/16

5 To run the post-read of the plate: Open the pre- read allelic discrimination plate document. Click instrument tab and select the post read button. The result will show the pre-read data subtracted from the post-read data for the run. The table below shows the correlation between fluorescence signals and sequences present in the samples. Fluorescence signals and allelic content a substantial increase in.. indicates.. FAM Fluorescense Only VIC Fluorescense Only Both fluorescent Signals Wild type Homozygosity Mutation Homozygosity Heterozygosity Kit description The kit is designed for 96 reactions Sample Material Genomic DNA isolated from human blood. Kit Storage e Stability Store the unopened kit at 15 to 25 C through the expiration date printed on the label (6 months from the date of manufacture). Protect the amplification mix (Blu Cap) from light. Kit Performance Quality control Allelic discrimination may be done in the allelic discrimination window by comparison between samples and positive controls. The kit is function tested using the control template (Red Cap) provided whit the kit, according to the protocol described below. Specificity The FV real time Amplification mix is sequence specific for the human gene of Factor V. ST pag.12/16 ST pag.5/16

6 Kit Contents Select the spectra tab to make sure there was signal generated. The kit contains the following items: n Vials Label Content 4 vials (500 µl) 1 vial (15 µl) 4 vials (10 µl) 4 vials (10 µl) 4 vials (10 µl) 2 vials (20 µl) Factor V amplification mix Taq Polymerase Factor V positive control (wild-type allele) Factor V positive control (mutated allele) Factor V positive control (heterozygous) NTC FV Forward Primer, FV Reverse Primer, dntps, Passive reference, MGB Taqman FAM Probe, MGB Taqman VIC Probe, specific Buffer. Taq Polymerase Cloned Wild type Factor V gene for mutation G1691A Cloned Mutated Factor V gene for mutation G1691A Cloned Heterozygoos Factor V gene for mutation G1691A Negative Materials Required but Not Supplied MicroAmp Optical 96-Well Reaction Plate (Applied Biosystem - P/N N ) ABI PRISM Optical Adhesive Cover (Applied Biosystem - P/N ) MicroAmp Optical Tubes (Applied Biosystems - P/N N ) To perform a post read allelic discrimination assay, the plate must be thermal cycled. Termal cycling can be done on the ABI PRISM 7000 Sequence Detection System or another instrument. ST pag.6/16 ST pag.11/16

7 Overview This Chapter describes how to acquire and analyse the sample results with the ABI PRISM 7000 Sequenze detection system. Read the specific instruction manual for the analysis with other instruments. The analysis consist in two steps: Pre read: before amplification After read: post amplification Pre Read (Facultative) If you want to do a pre read of a plate document, you must run it before running the plate through the termal cycler. With this operation you can monitor the initial fluorescence for all the samples. With the allelic discrimination plate document still open, click instrument tab and select the pre-read button after checking the sample volume. The pre-read will take about one minut to complete. Procedure Page Preparation of the master mix 8 Amplification mix 8 2 Introduction ST pag.10/16 ST pag.7/16

8 This section describes how to prepare the the REAL-TIME PCR sample for the reactions of allelic discrimination. Preparation of the master mix Depending on the total number of reactions, prepare the MicroAmp Optical 96-Well Reaction or Optical Tubes. The samples and the standard controls must be amplify in double Prepare a master mix by multiplying the amount in the Volume column by the double of the number of the reactions to be run, plus 8 additional reactions for the standard and negative controls. Procede as described below for a 25 standard reactions. In a 1.5 ml reaction tube, add the following components: Mix gently Item Volume (µl/sample) FV Amplification mix 19,75 Enzyme mix 0,25 Total 20 Pipet 20 µl master mix to the tube or well Add 5 µl of extracted -DNA from human whole blood or 5 µl negative control In different positions, add 5 µl of each dilutions of the standard controls. Seal the plate with optical adhesive cover or the tubes with optical caps. Place the plate or the tubes in the ABI PRISM Sequence Detection System Cycle the samples as described in the following section Hold 35 Cycles Pre-denaturation Denaturation Annealing-Extension T( C) Time T( C) Time T( C) Time 95 C C C 1 Volume 25 µl Data interpretation Page Overview 10 Pre read 10 Post read 12 Allelic Discrimination 13 3 ST pag.8/16 ST pag.9/16