Maize Bt-176 quantification

Transcription

1 Primerdesign Ltd GMO event quantification Maize Bt-176 quantification Detection and quantification of GMO integration events by real-time PCR GMO screening Event specific detection by real-time PCR

2 Contents Introduction 3 Kit Contents 5 Reagents and Equipment to Be Supplied by User 5 Kit Storage 5 Suitable Sample Material 5 Licensing Agreement and Limitations of Use 6 Primerdesign Satisfaction Guarantee 6 Quality Control 6 Bench-side Protocol 7 Amplification Protocol 9 Interpretation of Results 10 2

3 Introduction Event 176 was produced by transformation of the inbred line CG00526 with two plasmids. One plasmid contained two copies of a 3 truncated cry1ab gene, each regulated by different promoter sequences. The cry1ab open reading frame, corresponding to the sequence encoding the N-terminal 648 amino acids of the native Cry1Ab protein, was modified for optimal expression in plant cells. Green tissue expression of one copy of the cry1ab gene was regulated by the phosphoenolpyruvate carboxylase promoter while expression of the other cry1ab gene was controlled by a pollen specific promoter isolated from maize. Both genes employed 3 -polyadenylation sequences from the 35S transcript of cauliflower mosaic virus (CaMV). The Bt-maize Event 176 (tradenames NaturGard KnockOut ) was developed through a specific genetic modification to be resistant to attack by European corn borer (ECB; Ostrinia nubilalis), a major insect pest of maize in agriculture. These novel plants produce a truncated version of the insecticidal protein, Cry1Ab, derived from Bacillus thuringiensis subp. kurstaki strain HD-1. Event 176 is also genetically modified to express the bar gene cloned from the soil bacterium Streptomyces hygroscopicus, which encodes a phosphinothricin-nacetyltransferase (PAT) enzyme. The PAT enzyme is useful as a selection marker enabling identification of transformed plant cells as well as a source of resistance to the herbicide phosphinothricin (also known as glufosinate ammonium, the active ingredient in the herbicides Basta, Rely, Finale, and Liberty Principals of the test This kit provides a method for detecting gene insertion events by real-time PCR. The kit is based on the PCR amplification and detection across the integration site of the inserted genetic marker. This DNA sequence is artificial and therefore only detectable in a genetically modified organism (GMO) containing that specific modification. Amplification is detected by means of a double dye probe (Taqman probe) which is degraded during PCR producing fluorescence. The fluorescence trace can be used to both detect and quantify the proportion of genetically modified DNA present in the sample. As well as primers and probe that detect the insertion event, the kit also contains primers and probe to detect the wild-type sequence. Also included is a control DNA containing GM DNA at a ratio of 1 part in 100 (1% GMO). This enables the proportion of GM DNA present in any given sample to be calculated. GMO Event specific primer and probes 3

4 The kit provides event specific primers and probe that detect a GM insertion event. The primers and probe are read through the FAM channel and detect the presence of the precise DNA modification event under test and no others. Wild-type control primers and probe The kit also provides primers and probe to detect the endogenous wild-type DNA of the host plant. These primers and probe are also detected through the FAM channel and are tested in a separate well. Detection of the wild-type DNA indicates that the extraction process has been successful. With detection levels of the wild-type sequence enabling calculation of the proportion of modified DNA included in the sample. This is critical for determining the % of GM DNA in mixed populations of plants. Positive control for Bt-176 and Maize sequences The kit provides a positive control template for both primer and probes sets. The positive control enables the user to obtain positive traces thereby proving that the PCR reactions have been set up and run correctly. A positive control for each primer set should be included each time a run is performed. The PCR amplicons for the two primer sets have been cloned into the same vector so that the same positive control template can be used for both primer sets. GMO reference control sample (crushed seeds) The kit contains crushed wild-type seeds which have been spiked with GMO seeds at a level of 1%. The DNA extracted from this sample is a control for the extraction process to show that DNA can be successfully extracted using your extraction protocol. Data from this control sample for the wild-type primer set and event specific primer set can be used to calculate the percentage of GM DNA present in test samples. 4

5 Kit Contents Maize-WT DNA primer/probe mix (100 reactions BROWN) Bt-176 (GMO) DNA primer/probe mix (100 reactions BROWN) Positive control template (RED) GMO reference control sample (BLUE) RNAse/DNAse free water Reagents and Equipment to Be Supplied by User Real-Time PCR Instrument Mastermix or Mastermix components This kit is designed to work well with all commercially available Mastermixes. However, we recommend the use of Primerdesign 2x PrecisionPlus TM Mastermix. Pipettes and Tips Vortex and centrifuge Thin walled 0.2 ml PCR reaction tubes Kit Storage This kit is stable at room temperature but should be stored at -20ºC on arrival. Freeze/thawing cycles should be kept to a minimum once resuspended. Under these conditions reagents are stable for six months from date of resuspension. Suitable Sample Material All kinds of sample material suited for PCR amplification can be used. Please ensure the samples are suitable in terms of purity, concentration, and DNA integrity. Always run at least one negative control with the samples. To prepare a negative control, replace test sample DNA with RNAse/DNAse free water. 5

6 Licensing Agreement and Limitations of Use PCR is covered by several patents owned by Hoffman-Roche Inc and Hoffman-LaRoche, Ltd. Purchase of Primerdesign kits does not include or provide licence with respect to any patents owned by Hoffman-La Roche or others. Primerdesign Satisfaction Guarantee Primerdesign takes pride in the quality of all our products. Should this product fail to perform satisfactorily when used according to the protocols in this manual, Primerdesign will replace the item free of charge. Quality Control As part of our ISO9001 and ISO13485 quality assurance system, all Primerdesign products are monitored to ensure the highest levels of performance and reliability. 6

7 Bench-side Protocol To minimise the risk of contamination with foreign DNA, we recommend that all pipetting should be performed in a PCR clean environment. Ideally this would be a designated PCR cabinet. Filter tips are recommended for all pipetting steps. The positive control template is a significant contamination risk and should therefore be pipetted after the negative control and sample wells. 1. Pulse-spin each tube in a centrifuge before opening This will ensure the lyophilized primer and probe mix is in the base of the tube and is not spilt upon opening the tube. 2. Resuspend lyophilised primer and probe mix in the RNAse/DNAse free water provided. To ensure complete resuspension, vortex each primer and probe mix thoroughly, allow to stand for 5 minutes and vortex again before use. Component Maize Primer/Probe mix (BROWN) Bt-176 Primer/Probe mix (BROWN) Foil wrapped Positive control template (RED) * Volume 110 μl 110 μl 500 μl * This component contains high copy number template DNA and is a significant contamination risk. It must be opened and handled in a separate laboratory environment, away from the other components. There is a 10% over pipette in each of the primer/probe kits 3. Perform DNA extraction on tests samples and crushed seeds The crushed seeds should be extracted in parallel and using the same protocol as for all of the samples under test. N.B. the kit contains the correct amount of seeds required for 1 extraction only 7

8 4. Prepare complete reaction mixes for the Maize and Bt-176 primers Reaction mixes should be made for both the Maize and Bt-176 primers. Make up sufficient reactions for each primer and probe set to complete the following tests. 1 reaction for each sample, 1 reaction for the extracted crushed seeds 1 reaction as a no template control (NTC) 1 reaction for the positive control Component Primerdesign 2X PrecisionPlus TM Mastermix primer and probe mix Maize or Bt-176 (BROWN) RNAse/DNAse free water Final volume 1 reaction 10 μl 1 μl 4 μl 15 μl 5. Dispense 15ul of each Mastermix according to your plate layout 6. Dispense 5l of extracted sample DNA according to your plate set up To obtain a strong signal, the ideal concentration of DNA is 1-3ng/l. The concentration should not exceed 5ng/l. Substitute sample DNA for RNase/DNase free water as a negative control 7. Dispense 5l of extracted control DNA (crushed seeds) according to your plate set up To obtain a strong signal, the ideal concentration of DNA is 1-3ng/l. The concentration should not exceed 5ng/l. 8. Dispense 5l of positive control DNA (RED) according to your plate set up Positive control reactions should be included for both primer and probe sets each time the kit is used. The positive control contains the DNA for both primer and probe sets 8

9 Amplification Protocol 1. Amplification conditions using PrecisionPlus TM Mastermix Cycling x40 Step Time Temp Enzyme activation 2min 95ºC Denaturation 10s 95ºC DATA COLLECTION* 60s 60ºC *Fluorogenic data should be collected during this step through the FAM channel. 9

10 Interpretation of Results The presence of the GMO sequence within a sample can be calculated in percentage terms by comparison of the test sample results to the reference control results. Using the delta Ct method and normalising for experimentally derived errors, the GMO content of the test sample can be simply calculated. Sample data and calculations 1) Relative expression for test samples is calculated using the delta CT (cycle threshold) method and converted to a percentage. Test sample GMO event target CT = 29 WT event target CT = 25 Percentage GMO = (2^ - (29 25)) x 100 = 6.25%. 2) Relative expression for 1% reference control is calculated using the delta CT (cycle threshold) method and converted to a percentage. 1% GMO control sample (supplied) GMO event target CT = 32 WT event target CT = 26.5 Percentage GMO = (2^ - ( )) x 100 = 2.2 % 3) Calculate the normalising factor value (K) In an ideal situation the calculated GMO reference control content should have been 1%. However due to variation between experimental conditions and different hardware platforms this value can differ. The GMO 1% data is used as a normalising factor to correct for any systemic errors in the experiment. Normalising factor (K) = 1/experimental GMO 1% proportion K = 1% / 2.2 % = ) Correct the test sample data using the normalising factor (K) Final sample GMO content = measured GMO percentage x K Test sample percentage = 6.25 % x 0.45 = 2.81% 10