WO2005021785A1

WO2005021785A1 - Standards in pcr

Info

Publication number: WO2005021785A1
Application number: PCT/GB2004/003644
Authority: WO
Inventors: Tsuyako Katrina Ohara; Peter Bowler
Original assignee: Rhm Technology Limited
Priority date: 2003-08-29
Filing date: 2004-08-27
Publication date: 2005-03-10
Also published as: GB0320329D0; GB2405405A

Abstract

The invention provides the use of a standard in a method of nucleic acid sequence-based quantification on a mixture comprising a plurality of constituents, each constituent comprising a common nucleotide sequence, and a constituent of interest having a specific nucleotide sequence, characterised in that the standard is prepared from single said constituent comprising the said common sequence and a constituent-specific sequence in a known or elucidatable fixed copy number ratio.

Description

STANDARDS L PCR This invention relates to techniques for establishing standards for the characterization/quantification of genetically distinct constituents. Molecular methods are able to provide a useful and selective means for the quantification of one biological material present in admixture with other closely related biological materials. This is possible due to the presence of unique DNA sequences in all species. If the sequences have been characterized, then they can be detected selectively and quantified. Currently the method of choice involves use of the polymerase chain reaction (PCR) in which targeted sequences of DNA are amplified. The amount can be quantified by means of a modified PCR technique, as described below. Various chemistries are available for the quantification, but all are based upon binding of a modified DNA sequence (a probe) to the target strands, which fluoresces only when degraded or modified as the target sequences are amplified. This fluorescence is detected as a threshold cycle value (Ct), the threshold being the point at which sample fluorescence can be detected above the inherent background noise during amplification. Quantification is based upon measurement of a specific DNA sequence present only in the "adulterating" material and comparing its fluorescent signal with one similarly obtained from a "marker gene" present in both the

"adulterating" and "adulterated" materials. There are two commonly used techniques for achieving this. The first involves using a range of comparative standards. The Ct value for the sequence of interest is subtracted from that for the marker gene and the difference is compared to similar data obtained from a series of standards. This method is known as the Delta Ct (ΔCt) approach. The second uses a single standard (in contrast to the range used above) which is itself diluted to produce a range of target DNA concentrations. The

Ct values obtained for each of the "adulterating" and marker gene sequences are then compared with the equivalents for the diluted standard from which the relative number of copies of DNA present may be determined for the unknown sample. The ratio of these copy numbers for "adulterant" and marker gene sequences gives the proportion of adulterant. This is known as the copy number approach. For the avoidance of doubt, the term "copy number" as used herein is intended to mean a mathematically derived figure used to compare proportions of sequences of interest. Currently, quantification using PCR requires the use of prepared standards as a calibrant. These standards may be those produced commercially, e.g. for Roundup Ready Soya™ (RRS), 0.1 - 5% RRS in normal soya used in the Δ Ct method , or a single prepared standard used over a range of dilutions used for the copy number approach. Currently, both approaches rely upon blends of 0% and 100% of materials of interest to produce a range of standards for use. Such standards are prepared and distributed by specialist organisations, such as IRMM (Institute for Reference Materials and Measurements) in Europe, and are known as Certified Reference Materials. The major disadvantage of this approach is that by blending two biological materials, errors and uncertainties arise due to the inherent variability of the biological materials themselves. The absolute levels of components contained in such biological materials will be affected by their growth environment, their variety and a number of other factors and hence the levels of specific components will vary. Consequently, the relative amounts of materials of interest, such as DNA, may not be in the same ratios as the weights of components, even in accurately weighed blends. It is an object of the present invention to seek to mitigate these problems. According to the invention there is provided the use of a standard in a method of nucleic acid sequence-based quantification on a mixture comprising a plurality of constituents, each constituent comprising a common nucleotide sequence, and a constituent of interest having a specific nucleotide sequence, characterised in that the standard is prepared from a single said constituent comprising the said common sequence and a constituent-specific sequence in a known or elucidatable fixed copy number ratio. The standard may be any individual single variety microsample, such as a single seed or part thereof or like specimen of a vegetable variety. Alternatively it could be from a monoculture of an organism or from tissue of an animal species. As will be appreciated, the actual type or source of nucleic acid is not important as long as the requirement is satisfied that the constituent of interest comprises a common sequence and a constituent-specific sequence in a known or elucidatable fixed copy number ratio. Thus, standards according to the invention have an extremely broad utility. The method may for example be a DNA-based method of quantification such as, for example, PCR. However it will be appreciated that the standards will find utility in any DNA-based quantification technique. According to a second aspect of the invention there is provided a method for preparing a standard for use in a method of nucleic acid sequence- based quantification on a mixture comprising a plurality of constituents, each constituent comprising a common nucleotide sequence and a constituent of interest having a specific nucleotide sequence, the method characterised by the step of preparing the standard from a single said constituent comprising the said common sequence and a constituent-specific sequence in a known or elucidatable, fixed copy number ratio. According to a third aspect of the invention there is provided a standard for use in a method of nucleic acid sequence-based quantification on a mixture comprising a plurality of constituents, each constituent comprising a common nucleotide sequence and a constituent of interest having a specific nucleotide sequence, characterised in that the standard comprises a single said constituent comprising the said common sequence and a constituent-specific sequence in a known or elucidatable fixed copy number ration. The present invention has been devised in association with a desire to develop a pasta standard for PCR. Such a standard is necessary in order to establish clear limitation on the quantities of breadmaking or other flour that may be incorporated into a mix with durum flour for the manufacture of pasta products, and for monitoring compliance with regulations governing those limitations. However, far broader implications have since been envisaged and recognised. By an individual single variety microsample, there is to be understood in the context of the present description a single seed or like specimen of a vegetable variety; or a microsample from a monoculture of an organism such as bacteria; or a microsample of tissue from an animal species, which will be genetically unique to its variety yet capable of representing a standard for the genetic content of the variety. The microsample should be tested for the presence of a sequence of interest (the "adulterant") and, if that sequence is present, then that microsample by definition represents an absolute standard and may be used with total confidence as the standard for copy number approach PCR or other DNA-based quantification techniques. As stated above, the invention has been derived from a consideration of the problems associated with the preparation of pasta standards for PCR, namely the effects of variety, grain filling and other variables, all of which affect the ratios of DNA which can be extracted in standard blends in T. aestivum and T. durum. Working with the copy number approach to PCR it has been possible to use nominally 100% product as a standard successfully for quantifications. It is apparent that this concept can be applied generically to PCR and to other

DNA-based amplification techniques, where both the specific gene or sequence to be measured (the "adulterant") and the normalising or marker gene or sequences are present in the material to be quantified, i.e. the standard is 100% of the "adulterating" sequence. If these genes or sequences are present as single copies or in known numbers (rather than variable) then such materials can provide absolute standards for copy number approach PCR or other DNA based qualification techniques. The basis of the invention is therefore the use of only one material as a source of both "adulterant" and "normalising" genes or sequences with fixed copy number ratios, whereby the ratio between these is absolute, i.e. it cannot vary as in blended materials. To ensure unequivocally that the standard is absolute, a single seed or part thereof or part of a single organism should be used. The seed should first be tested for the presence of the "adulterating" sequence. If that sequence is present, then the seed by definition represents an absolute standard, and can be used as the absolute standard for the copy number approach PCR or other appropriate methodologies. The use of single seeds, or single organism material, obviates the need for conventional certified standards as the material may be self-certified by a qualitative check for the presence of the "adulterating" sequence. Any T. aestivum grain could be used as a standard for the quantification of T. durum adulterated by T. aestivum. In this grain, there will be in the D genome (present only in T. aestivum) a specific sequence that can be measured in order to determine aestivum content, and the normalising A/B genomes present in both aestivum and durum. Hence, any single T. aestivum grain of any variety or quality may be used as an absolute standard. In the copy number approach to PCR, DNA is extracted and diluted serially to provide relative amounts of each of these DNA sequences, and samples may be measured for composition against each of the D and A/B genome sequences. From the dilution of the standard which is equivalent to the A/B and D genomes concentration in the sample, the percentage of T. aestivum can be readily calculated. The benefit of this is that ranges of standards containing both T. aestivum and T. durum are not required. EXPERIMENT TO VALIDATE THE USE OF LEAF FROM ANY GERMINATED SINGLE T. AESTIVUM GRAIN AS STANDARD To confirm or validate that any T. aestivum grain could be used as a standard for the quantification of T. durum adulterated by T. aestivum the following validation experiment was performed. Five geographically representative (UK, North America and Europe) varieties of T. aestivum grains were selected; Hereward (UK), Laura (USA), Marton Vasari 19 (Hungary), Ritmo (Holland) and GK Zombar (Hungary).

These grains were allowed to germinate and subsequently planted into labelled pots of compost. For each of the selected variety, a leaf from 8 distinct grains were individually ground using Fastprep FP120 (Buffer, glass balls, acid washed sand and cut leaf) and the DNA was extracted and purified. Concentration of the extracted DNA was measured by fluorescence using Sigma DNA Quantitation Kit, and where necessary DNA extracts were diluted to the concentration of 20ng/ul. The DNA extracts and extraction blank were analysed for the presence of T. aestivum (final maximum DNA cone, in PCR = lOOng). The extraction blanks were required not to contain T. aestivum, and in order the carry out this statistically designed experiment, minimum of six confirmed T. aestivum DNA extracts from each of the four varieties were required. Hereward, Laura, Marton Vasari 19 and Ritmo leaves were used in the following experiment. Having randomly selected six T. aestivum positive extracts per variety, the remaining extracts were set aside. The composite stock standard (enough to run six PCR plates) was made by taking equal volumes (30ul) from each of the 24 concentration adjusted DNA extracts. Six randomised plates of 3x3 5ul quantitative PCR according to the table below (fig: 1 & 2) were carried out using the serially diluted composite standards and data was calculated using relative copy number. All of the DNA extracts (except standards) were further diluted x4 for PCR to ensure that they fell within the range of the standards. Singleplex real-time PCR was carried out on a single ABI 7700, for the D-genome (FAM labelled) and the normaliser (TET labelled) over minimum time in order to reduce further variables.

Hereward: Originl Laura: Origin 2 Marton Vasari 19: Origin 3 Ritmo: Origin 4

Fig:l Statistically designed randomised experimental design

Fig: 2 Example of PCR plate layout of plate one 2:6:1 denotes Origin:Seed:Replicate Results

Statistical analysis Model 1 General Linear Model: Result versus Plate, Origin, seed Source DF Seq SS Adj SS Adj MS F P Plate 5 7801.4 8739.7 1747.9 1 99 0 105 Origin 3 606.5 563.8 187.9 0 21 0 886 seed 5 805.0 805.0 161.0 0 18 0 967 Error 34 29797.4 29797.4 876.4 Total 47 39010.4 Unusual Observations for Result Obs Result Fit SE Fit Residual St Resid 47 244.400 125.707 14.665 118.693 4.62R R denotes an observation with a large standardized residual .

Model 2 General Linear Model: Result_l versus Plate, Origin, seed Source DF Seq SS Adj SS Adj MS F P Plate 5 6581.3 6819.7 1363.9 3.96 0 006 Origin 3 2807.1 2712.7 904.2 2.62 0 066 seed 5 812.8 812.8 162.6 0.47 0 795 Error 34 11714.9 11714.9 344.6 Total 47 21916.1 Unusual Observations for Result 1 Obs Result 1 Fit SE Fit Residual St Resid 3 165.300 130 021 9 526 35.279 2.21R 8 78.600 112 412 10 423 -33.812 -2.20R 21 135.900 99 589 9 872 36.311 2.31R R denotes an observation with a large standardized residual.

Model 3 General Linear Model: Result 1 versus Plate, Origin Analysis of Variance for Result_l, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Plate 5 6581.3 7169.8 1434.0 4.46 0.003 Origin 3 2807.1 2807.1 935.7 2.91 0.046

Error 39 12527.7 12527.7 321.2

Total 47 21916.1

Unusual Observations for Result 1

Obs Result 1 Fit SE Fit Residual St Resid 3 165.300 131. .874 7. .440 33.426 2.05R 8 78.600 111. .658 8. .484 -33.058 -2.09R 21 135.900 95, .197 7. .973 40.703 2.54R

Main Effects Plot - LS Means for Result 1

Using all the results provided, there are no significant differences between origin, seeds or plates (Model 1 above). However, this result is strongly influenced by a very big outlier (observation 4, result 244.4). On close examination of the cycle thresholds (Cts) generated during PCR, there is no technical reason for dismissing the data, and as such the conclusion based on Model one is deemed sound. However, our statistician has also provided Models 2 and 3 below. There is no technical reason for suspecting the results, but two other models substituting the outlier with the mean result value for all results (mean = 115) have also been produced. Model 2 shows that there is a very significant plate to plate difference, no seed to seed difference and a borderline non significant difference (at the 95% confidence level) between the origins. It would be significant at the 93.4% level. Since there are no differences between seeds this can be assumed to be just another contribution to the overall random variation. As a result, the model can be collapsed and the effects of plate and origin only (model 3) may be considered. The conclusion here is that there is a very significant plate to plate difference and a borderline but this time significant difference (at the

95% confidence level) between the origins. Origin 3 gives a lower result than the rest. While this is statistically significant it may not be practically important.

Results of PCR - all values expressed as cycle thresholds (Cts)

Plate 1 Plate 2 mean mean mean mean D-genome D-genome Normaliser Normaliser D-genome D-genome Normaliser Normaliser

Sample Ct FA Ct FAM Ct TET Ct TET Sample CtFAM Ct FAM Ct TET Ct TET ntc 45 4500 45 4500 ntc 45 4500 45 4500 45 45 45 45 45 45 45 45 std / 2 2413 2393 2347 2353 std/ 2 2381 2399 2351 2332 2411 237 2412 233 2356 2343 2403 2315 std / 4 2467 2469 2457 2467 std/4 248 2474 2439 2444 2492 248 246 2445 2448 2464 2483 2449 std / 8 2536 2535 2573 2561 std/ 8 2583 2575 252 2525 2539 2559 2556 2534 253 2552 2585 2522 std / 16 2702 2682 2679 2668 std/ 16 2665 2677 263 2633 2695 2655 2702 2654 2649 2669 2664 2615 std / 32 2791 2773 2733 2765 std / 32 2782 2819 2738 2745 2759 2782 2827 2773 2769 278 2849 2725 std / 64 2872 2869 2877 2884 std/ 64 2896 2909 2846 2863 2869 2899 2913 2859 2865 2877 2917 2883 std / std/ 128 2916 2965 2973 2983 128 2967 2990 295 2933 3008 3001 3002 2922 2971 2976 3002 2928 2 6 1 2463 2456 2466 2478 311 2505 2512 2434 2453 2452 2494 2501 2463 2454 2474 2529 2462 1 5 2 2442 2440 2459 2447 341 2494 2481 2439 2422 2447 2423 249 2436 2432 2458 246 2392 1 4 2 2418 2432 2473 2462 462 2447 2467 2442 2447 2456 246 2483 2471 2421 2453 2471 2429 4 5 2 2471 2457 2455 2459 211 2472 2493 2448 2474 2441 2481 2492 2439 246 2441 2516 2536 2 4 2 2461 2462 2467 2461 342 2461 2462 2435 2436 2446 2459 2463 2418 2478 2458 2462 2456 4 2 1 2464 2455 2462 2458 251 2457 2428 2431 2421 2439 244 2384 2418 2462 2472 2444 2413 131 2434 2449 2465 2475 151 2502 2490 2444 2460 2458 2471 2498 2444 2454 249 2471 2492 361 2501 2500 2498 2463 362 2478 2490 2465 2451 2504 2437 2503 2444 2494 2454 2489 2444

Results of PCR - all values expressed as cycle thresholds (Cts)

Plate 3 Plate 4 mean mean mean mean D-genome D-genome Normaliser Normaliser D-genome D-genome Normaliser Normaliser

Sample CtFAM CtFAM CtTET CtTET Sample CtFAM CtFAM CtTET CtTET ntc 45 4500 45 4500 ntc 45 4500 45 4500 45 45 45 45 45 45 45 45 std/ 2 2364 2370 2334 2320 std/ 2 241 2398 2309 2320 237 2308 2375 2335 2377 2319 2408 2317 std/4 2421 2452 2402 2415 std/4 2458 2470 2451 2422 2449 2413 2465 2413 2485 2429 2486 2403 std/ 8 2604 2563 2517 2508 std/ 8 2588 2586 2531 2506 2596 2518 2586 2515 249 249 2585 2471 std/ 16 2679 2679 2591 2598 std/ 16 2669 2680 2606 2614 271 2633 2684 2609 2649 257 2688 2626 std / 32 2788 2791 2707 2702 std / 32 2762 2794 2721 2716 2772 2691 2806 2729 2813 2707 2813 2698 std/ 64 2918 2902 2835 2836 std/ 64 29 2916 2809 2822 2922 2846 2918 2838 2866 2828 2931 282 std/ std/ 128 304 3028 293 2924 128 3015 2986 2916 2921 3008 2964 2945 2947 3036 2877 2998 2899 442 2473 2472 2401 2413 411 2418 2452 2399 2412 2464 2409 2459 2419 248 243 2479 2419 221 2467 2489 2412 2409 121 2454 2442 2375 2384 2503 2411 2428 2395 2496 2403 2444 2381 112 2492 2481 2403 2434 441 2465 2493 2415 2433 2482 2438 2506 2444 2468 246 2508 2439 332 2418 2484 2422 2407 241 2477 2477 2427 2407 2523 2389 2461 2396 251 2411 2493 2399 312 2414 2458 2446 2428 461 2462 2471 2421 2426 2485 2426 248 2426 2474 2413 2472 2432 232 249 2453 2417 2406 451 2453 2463 2427 2398 2439 2405 2459 2401 243 2396 2476 2366 432 2443 2469 2419 2434 262 2469 2478 2413 2414 2505 2427 2491 24 2459 2457 2475 2428 111 2458 2480 2437 2434 231 2512 2490 243 2418 2484 2427 2492 2398 2499 2438 2466 2427

Results of PCR - all values expressed as cycle thresholds (Cts)

Plate 5 Plate 6 mean mean mean mean D-genome D-genome Normaliser Normaliser D-genome D-genome Normaliser Normaliser

Sample CtFAM CtFAM CtTET CtTET Sample CtFAM CtFAM CtTET CtTET ntc 45 4500 45 4500 ntc 45 4500 45 4500 45 45 45 45 45 45 45 45 std/ 2 2411 2390 2314 2315 std/ 2 2405 2409 2342 2343 2386 2291 241 2344 2373 234 2413 2342 std/4 2467 2458 2413 2420 std/4 2523 2520 242 2432 2436 2437 2519 2456 247 2409 2518 2421 std/ 8 2575 2589 2519 2504 std/ 8 2607 2603 2505 2517 2586 2506 2594 2507 2607 2488 2608 2539 std/ 16 2639 2662 2621 2625 std / 16 2711 2712 2638 2641 2643 2602 2719 2651 2705 2653 2707 2634 std / 32 2813 2788 2675 2693 std / 32 2803 2802 2715 2718 2781 271 28 2725 2769 2694 2804 2713 std / 64 2917 2873 2824 2821 std / 64 2928 2918 2837 2844 2837 2823 2907 286 2866 2815 2919 2836 std/ std/ 128 2948 2979 2873 2891 128 2993 3009 2953 2943 2972 2885 2996 2949 3016 2914 3039 2926 162 2415 2457 2388 2406 161 2531 2516 2432 2438 2453 2411 2507 2439 2502 2419 2509 2442 331 2466 2479 2375 2393 222 2514 2510 2435 2430 2481 2406 2519 2431 2489 2398 2497 2425 1:2:2 24.67 24.82 24.19 23.91 3:2:1 25.23 25.22 24.45 24.51 25.03 23.78 25.25 24.52 24.77 23.76 25.18 24.57 2:1 :2 24.63 24.73 24.16 24.15 1 :4:1 25 24.98 24.45 24.29 24.76 24.16 24.87 24.23 24.8 24.14 25.06 24.18 2:5:2 24.54 24.44 23.5 23.81 1 :3:2 25.15 24.96 24.34 24.34 24.45 23.97 24.95 24.32 24.33 23.95 24.77 24.35 4:1 :2 24.73 24.91 24.16 24.10 4:3:1 24.73 24.95 24.31 24.36 25.12 24.03 25.04 24.38 24.88 24.11 25.07 24.4 4:2:2 24.55 24.56 23.77 23.98 3:2:2 24.25 24.72 24.76 24.66 24.59 24.15 24.86 24.58 24.54 24.01 25.06 24.64 3:5:2 24.73 24.90 24.32 24.09 3:5:1 25.13 25.12 24.25 24.36 24.87 23.88 25.1 24.41 25.11 24.08 25.13 24.42

Calculations

Using the formula below, the relative copy numbers for the standards and unknowns were calculated.

R = relative copy number ratio Normaliser : durum L 4 copies

A = % T. aestivum aestivum 6 copies D-genome : aestivum 2 copies

Therefore R =(((100-A) x 4) + (6A))/ 2 A A = 200 > / (R-l)

PCR Plate 1 mean mean relative log relative D-genome relative log relative i normaliser Sample 'copy' no 'copy' no Ct FAM 'copy' no 'copy' no Ct TET std / 2 640 2.81 23.93 1920 3.28 23.53 std / 4 320 2.51 24.69 960 2.98 24.67 std / 8 160 2.20 25.35 480 2.68 25.61 std / 16 80 1 .90 26.82 240 2.38 26.68 std / 32 40 1 .60 27.73 120 2.08 27.65 std / 64 20 1.30 28.69 60 1.78 28.84 std / 128 10 1 .00 29.65 30 1.48 29.83

Two graphs were generated, one for D-genome and another for the normaliser, by plotting log relative copy number against Cts, and a line of best fit was drawn through the points.

The equations y = -3.2654x + 32.909 and y = -3.4742x + 34.958 were used to calculate the relative copy numbers of the unknowns from the Cts as shown below. mean calculated calculated mean calculated calculated log D-genome log relative relative Normaliser relative relative Ct FAM 'copy' no 'copy' no Ct TET 'copy' no 'copy' no 2:6:1 24.56 2.56 359.57 24.78 2.93 850.35 1 :5:2 24.40 2.60 402.52 24.47 3.02 1046.61 1 :4:2 24.32 2.63 427.88 24.62 2.98 945.47 4:5:2 24.57 2.55 357.05 24.59 2.98 964.46 2:4:2 24.62 2.54 346.30 24.61 2.98 949.66 4:2:1 24.55 2.56 362.97 24.58 2.99 970.87 1 :3:1 24.49 2.58 379.55 24.75 2.94 865.51 3:6:1 25.00 2.42 264.90 24.63 2.97 939.23

Using the calculated relative copy numbers above, relative copy number ratios were calculated. Using the formula stated above (A = 200 / (R-1) where A = % T. aestivum and R = relative copy number ratio), % T. aestivum were calculated from the relative copy number ratios. D-genome Normaliser calculated calculated calculated 'copy' no % 'copy' no 'copy' no ratio T. aestivum 2:6:1 359.57 850.35 2.36 146.5 1 :5:2 402.52 1046.61 2.60 125.0 1 :4:2 427.88 945.47 2.21 165.3 4:5:2 357.05 964.46 2.70 117.6 2:4:2 346.30 949.66 2.74 114.8 4:2:1 362.97 970.87 2.67 119.4 1 :3:1 379.55 865.51 2.28 156.2 3:6:1 264.90 939.23 3.55 78.6

PCR Plate 2 mean mean relative log relative D-genome relative log relative normaliser Sample 'copy' no 'copy' no Ct FAM 'copy' no 'copy' no Ct TET std / 2 640 2.81 23.99 1920 3.28 23.32 std / 4 320 2.51 24.74 960 2.98 24.44 std / 8 160 2.20 25.75 480 2.68 25.25 std / 16 80 1.90 26.77 240 2.38 26.33 std / 32 40 1.60 28.19 120 2.08 27.45 std / 64 20 1.30 29.09 60 1.78 28.63 std / 128 10 1.00 29.90 30 1.48 29.33 Two graphs were generated, one for D-genome and another for the normaliser, by plotting log relative copy number against Cts, and a line of best fit was drawn through the points.

The equations y = -3.4267x + 33.44 and y = -3.3939x + 34.472 were used to calculate the relative copy numbers of the unknowns from the Cts as shown below. mean calculated calculated mean calculated calculated D-genome log relative relative Normaliser log relative relative Ct FAM 'copy' no 'copy' no Ct TET 'copy' no 'copy' no 3:1 :1 25.12 2.43 268.51 24.53 2.93 849.91 3:4:1 24.81 2.52 329.22 24.22 3.02 1046.48 4:6:2 24.67 2.56 362.50 24.47 2.95 883.22 2:1 :1 24.93 2.48 303.71 24.74 2.87 735.39 3:4:2 24.62 2.57 374.89 24.36 2.98 951.66 2:5:1 24.28 2.67 470.06 24.21 3.02 1058.38 1 :5:1 24.90 2.49 309.90 24.60 2.91 810.49 3:6:2 24.90 2.49 310.59 24.51 2.94 861.52

Using the calculated relative copy numbers above, relative copy number ratios were calculated. Using the formula stated above (A = 200 / (R-1) where A = % T. aestivum and R = relative copy number ratio), % T. aestivum were calculated from the relative copy number ratios. D-genome Normaliser calculated calculated calculated 'copy' no % 'copy' no 'copy' no ratio T. aestivum 3:1 :1 268.51 849.91 3.17 92.4 3:4:1 329.22 1046.48 3.18 91.8 4:6:2 362.50 883.22 2.44 139.2 2:1 :1 303.71 735.39 2.42 140.7 3:4:2 374.89 951.66 2.54 130.0 2:5:1 470.06 1058.38 2.25 159.8 1 :5:1 309.90 810.49 2.62 123.8 3:6:2 310.59 861.52 2.77 112.8

PCR Plate 3 mean mean relative log relative D-genome relative log relative normaliser Sample 'copy' no 'copy' no Ct FAM 'copy' no 'copy' no Ct TET std / 2 640 2.81 23.70 1920 3.28 23.20 std / 4 320 2.51 24.52 960 2.98 24.15 std / 8 160 2.20 26.00 480 2.68 25.08 std / 16 80 1.90 26.79 240 2.38 25.98 std / 32 40 1.60 27.91 120 2.08 27.02 std / 64 20 1.30 29.02 60 1.78 28.36 std / 128 10 1.00 30.28 30 1.48 29.24 Two graphs were generated, one for D-genome and another for the normaliser, by plotting log relative copy number against Cts, and a line of best fit was drawn through the points.

The equations y = -3.6359x + 33.809 and y = -3.3773x + 34.186 were used to calculate the relative copy numbers of the unknowns from the Cts as shown below. mean calculated calculated mean calculated calculated log D-genome relative relative Normaliser log relative relative Ct FAM 'copy' no 'copy' no Ct TET 'copy' no 'copy' no 4:4:2 24.72 2.50 315.41 24.13 2.98 947.41 2:2:1 24.89 2.45 284.42 24.09 2.99 978.04 1 :1 :2 24.81 2.48 299.20 24.34 2.92 824.77 3:3:2 24.84 2.47 293.57 24.07 2.99 986.97 3:1 :2 24.58 2.54 346.11 24.28 2.93 855.31 2:3:2 24.53 2.55 356.49 24.06 3.00 995.99 4:3:2 24.69 2.51 322.14 24.34 2.91 821.03 1 :1 :1 24.80 2.48 299.83 24.34 2.92 822.90 Using the calculated relative copy numbers above, relative copy number ratios were calculated. Using the formula stated above (A = 200 / (R-1) where A = % T. aestivum and R = relative copy number ratio), % T. aestivum were calculated from the relative copy number ratios. D-genome Normaliser calculated calculated calculated 'copy' no % 'copy' no 'copy' no ratio T. aestivum 4:4:2 315.41 947.41 3.00 99.8 2:2:1 284.42 978.04 3.44 82.0 1 :1 :2 299.20 824.77 2.76 113.9 3:3:2 293.57 986.97 3.36 84.7 3:1 :2 346.11 855.31 2.47 135.9 2:3:2 356.49 995.99 2.79 111.5 4:3:2 322.14 821.03 2.55 129.1 1 :1 :1 299.83 822.90 2.74 114.6

Plate 4 mean mean relative log relative D-genome relative log relative normaliser Sample 'copy' no 'copy' no Ct FAM 'copy' no 'copy' no Ct TET std / 2 640 2.81 23.98 1920 3.28 23.20 std / 4 320 2.51 24.70 960 2.98 24.22 std / 8 160 2.20 25.86 480 2.68 25.06 std / 16 80 1.90 26.80 240 2.38 26.14 std / 32 40 1.60 27.94 120 2.08 27.16 std / 64 20 1.30 29.16 60 1.78 28.22 std / 128 10 1.00 29.86 30 1.48 29.21

D-genome standard Normaliser standard curve

The equations y = -3.3998x + 33.37 and y = -3.3354x + 34.112 were used to calculate the relative copy numbers of the unknowns from the Cts as shown below. mean calculated calculated mean calculated calculated D-genome log relative relative Normaliser log relative relative Ct FAM 'copy' no 'copy' no Ct TET 'copy' no 'copy' no 4:1 :1 24.52 2.60 400.95 24.12 2.99 987.97 1 :2:1 24.42 2.63 429.05 23.84 3.08 1204.18 4:4:1 24.93 2.48 303.74 24.33 2.93 858.58 2:4:1 24.77 2.53 338.50 24.07 3.01 1022.67 4:6:1 24.71 2.55 351.75 24.26 2.95 896.95 4:5:1 24.63 2.57 373.01 23.98 3.04 1090.73 2:6:2 24.78 2.53 335.46 24.14 2.99 978.92 2:3:1 24.90 2.49 309.97 24.18 2.98 947.88 Using the calculated relative copy numbers above, relative copy number ratios were calculated. Using the formula stated above (A = 200 / (R-1) where A = % T. aestivum and R = relative copy number ratio), % T. aestivum were calculated from the relative copy number ratios. D-genome Normaliser calculated calculated calculated 'copy' no % 'copy' no 'copy' no ratio T. aestivum 4:1 1 400.95 987.97 2.46 136.6 1 :2 1 429.05 1204.18 2.81 110.7 4:4 1 303.74 858.58 2.83 109.5 2:4 1 338.50 1022.67 3.02 99.0 4:6 1 351.75 896.95 2.55 129.0 4:5 1 373.01 1090.73 2.92 103.9 2:6:2 335.46 978.92 2.92 104.3 2:3:1 309.97 947.88 3.06 97.2

PCR Plate 5 mean mean relative log relative D-genome relative log relative normaliser Sample 'copy' no 'copy' no Ct FAM 'copy' no 'copy' no Ct TET std / 2 640 2.81 23.90 1920 3.28 23.15 std / 4 320 2.51 24.58 960 2.98 24.20 std / 8 160 2.20 25.89 480 2.68 25.04 std / 16 80 1.90 26.62 240 2.38 26.25 std / 32 40 1.60 27.88 120 2.08 26.93 std / 64 20 1.30 28.73 60 1.78 28.21 std / 128 10 1.00 29.79 30 1.48 28.91

The equations y = -3.3168x + 33.082 and y = -3.2242x + 33.772 were used to calculate the relative copy numbers of the unknowns from the Cts as shown below. mean calculated calculated mean calculated calculated D-genome log relative relative Normaliser log relative relative Ct FAM 'copy' no 'copy' no Ct TET 'copy' no 'copy' no 1 :6:2 24.57 2.57 369.26 24.06 3.01 1028.54 3:3:1 24.79 2.50 316.96 23.93 3.05 1128.60 1 :2:2 24.82 2.49 308.99 23.91 3.06 1144.84 2:1 :2 24.73 2.52 329.68 24.15 2.98 962.22 2:5:2 24.44 2.61 403.20 23.81 3.09 1232.52 4:1 :2 24.91 2.46 290.95 24.10 3.00 999.57 4:2:2 24.56 2.57 370.97 23.98 3.04 1091.61 3:5:2 24.90 2.47 292.30 24.09 3.00 1004.34

Using the calculated relative copy numbers above, relative copy number ratios were calculated. Using the formula stated above (A = 200 / (R-1) where A = % T. aestivum and R = relative copy number ratio), % T. aestivum were calculated from the relative copy number ratios.

D-genome Normaliser calculated calculated calculated 'copy' no % 'copy' no 'copy' no ratio T. aestivum 1 :6:2 369.26 1028.54 2.79 112.0 3:3:1 316.96 1128.60 3.56 78.1 1 :2:2 308.99 1144.84 3.71 73.9 2:1 :2 329.68 962.22 2.92 104.2 2:5:2 403.20 1232.52 3.06 97.2 4:1 :2 290.95 999.57 3.44 82.1 4:2:2 370.97 1091.61 2.94 103.0 3:5:2 292.30 1004.34 3.44 82.1

PCR Plate 6 mean mean relative log relative D-genome relative log relative normaliser

Sample 'copy' no ' copy' no Ct FAM 'copy' no 'copy' no Ct TET std / 2 640 2.81 24.09 1920 3.28 23.43 std / 4 320 2.51 25.20 960 2.98 24.32 std / 8 160 2.20 26.03 480 2.68 25.17 std / 16 80 1.90 27.12 240 2.38 26.41 std / 32 40 1.60 28.02 120 2.08 27.18 std / 64 20 1.30 29.18 60 1.78 28.44 std / 128 10 1.00 30.09 30 1.48 29.43 Two graphs were generated, one for D-genome and another for the normaliser, by plotting log relative copy number against Cts, and a line of best fit was drawn through the points.

The equations y = -3.3164x + 33.418 and y = -3.3512x + 34.316 were used to calculate the relative copy numbers of the unknowns from the Cts as shown below. mean calculated calculated mean calculated calculated D-genome log relative relative Normaliser log relative relative Ct FAM 'copy' no 'copy' no Ct TET 'copy' no 'copy' no 1 :6:1 25.16 2.49 309.78 24.38 2.97 924.49 2:2:2 25.10 2.51 322.21 24.30 2.99 972.27 3:2:1 25.22 2.47 296.45 24.51 2.93 841.63 1 :4:1 24.98 2.55 351.02 24.29 2.99 983.46 1 :3:2 24.96 2.55 355.93 24.34 2.98 950.25 4:3:1 24.95 2.55 358.41 24.36 2.97 933.00 3:2:2 24.72 2.62 418.52 24.66 2.88 760.95 3:5:1 25.12 2.50 317.77 24.36 2.97 935.14 Using the calculated relative copy numbers above, relative copy number ratios were calculated. Using the formula stated above (A = 200 / (R-1) where A = % T. aestivum and R = relative copy number ratio), % T. aestivum were calculated from the relative copy number ratios.

D-genome Normaliser calculated calculated calculated 'copy' no % 'copy' no 'copy' no ratio T. aestivum 1 :6:1 309.78 924.49 2.98 100.8 2:2:2 322.21 972.27 3.02 99.1 3:2:1 296.45 841.63 2.84 108.8 1 :4:1 351.02 983.46 2.80 111.0 1 :3:2 355.93 950.25 2.67 119.8 4:3:1 358.41 933.00 2.60 124.8 3:2:2 418.52 760.95 1.82 244.4 3:5:1 317.77 935.14 2.94 102.9 The following is presented as a practical example of the overall concept embodied by the invention.

EXAMPLE An experiment was performed to measure the T. aestivum content of a T. durum sample containing nominally 2% of T. aestivum. A T. aestivum grain was allowed to germinate and grow, and a leaf was harvested. The leaf was ground using Fastprep FP120 (Buffer, glass balls, acid washed sand and cut leaf). From the homogenous material of nominal 2% T. aestivum content, DNA was extracted from eight samples, together with the extraction blank and the leaf standard material using an appropriate extraction and clean up method. Concentration of the extracted DNA was measured by fluorescence using Sigma DNA Quantitation Kit, and where necessary DNA extracts were diluted to the concentration of 20ng/ul. The DNA extracts were qualitatively analysed for the presence of T. aestivum. The extraction blanks were required not to contain any T. aestivum. For the standards, starting with the DNA concentration of 20ng/ul, six levels of serial dilutions were performed (x3 dilutions) to obtain seven standards of differing concentrations. For the samples, DNA concentration was adjusted so that the cycle threshold (Ct) values would fall within the range of the standards (5ng/ul). Singleplex real-time PCR was carried out on ABI 7700, for the D- genome (FAM labelled) and the normaliser (TET labelled) with 3x3 5ul of DNA addition to the appropriate wells (fig.l).

Fig: 1 PCR plate layout

Results of PCR - all values expressed as cycle thresholds (Cts)

D-genome Normaliser mean mean

Sample Ct FAM Ct FAM Ct TET Ct TET ntc* 45 45.00 45 45.00 45 45 45 45 std/2 24.31 24.24 23.27 23.10 24.19 23.21 24.21 22.81 std 6 25.8 25.83 25.13 24.72 25.88 24.57 25.82 24.45 std 18 27.09 27.14 26.48 26.37 27.14 26.41 27.18 26.23 std/54 28.82 28.81 27.91 28.08 28.99 28.28 28.61 28.04 std/162 30.56 30.50 29.7 29.40 30.47 29.05 30.48 29.44 std/486 32.38 32.39 31.05 31.16 32.36 31.23 32.44 31.2 std/1458 34.73 34.19 32.57 32.68 33.62 32.78 34.22 34.43** unknown a 31.98 31.92 25.26 25.22 32.2 25.17 31.58 25.22 unknown b 31.97 31.92 25.18 25.27 32.13 25.22 31.66 25.42 unknown c 31.1 31.13 24.29 24.34 31.22 24.41 31.08 24.31 unknown d 32.09 31.55 25 24.88 31.08 24.96 31.47 24.68 unknown e 31.64 31.38 24.44 24.54 31.24 24.51 31.27 24.68 unknown f 31.7 31.58 25.3 25.29 31.37 25.19 31.66 25.38 unknown g 31.84 32.09 25.4 25.36 32.18 25.31 32.25 25.38 unknown h 32.2 32.18 25.54 25.45 32.02 25.37 32.32 25.45

* ntc = no template control

34.43** not used in calculation due to poor reproducibility

Calculation

Using the formula below, the relative copy numbers for the standards were calculated

R = relative copy number ratio Normaliser durum 4 copies A = % T. aestivum aestivum 6 copies D-genome : aestivum 2 copies

Therefore R =(((100- A) x 4) + (6A))/ 2A A = 200 / (R-1) D-genome Normaliser relative log relative mean relative log relative mean Sample 'copy' no 'copy' no Ct FAM 'copy' no 'copy' no Ct TET std/2 7290 3.86 24.24 21870 4.34 23.10 std/6 2430 3.39 25.83 7290 3.86 24.72 std/18 810 2.91 27.14 2430 3.39 26.37 std/54 270 2.43 28.81 810 2.91 28.08 std/162 90 1.95 30.50 270 2.43 29.40 std/486 30 1.48 32.39 90 1.95 31.16 std/1458 10 1.00 34.19 30 1.48 32.68

-3.46922X + 37.44922 R² = 0.99713

1.00 2.00 3.00 4.00 log relative copy num ber y = -3.34184x + 37.64756 R² = 0.99936

1.00 2.00 3.00 4.00 5.00 log relative copy num ber

The equations y = -3.46922x + 37.44922 and y = -3.34184x + 37.64756 were used to calculate the relative copy numbers of the unknowns from the Cts as shown below.

D-genome Calculated D-genome Normaliser Calculated Normaliser calculated calculated mean log relative relative mean log relative relative Ct FAM 'copy' no 'copy^* no Ct TET 'copy' no 'copy' no unknown a 31.92 1.59 39.25 25.22 3.72 5245.36 unknown b 31.92 1.59 39.25 25.27 3.70 5044.50 unknown c 31.13 1.82 66.15 24.34 3.98 9618.41 unknown d 31.55 1.70 50.28 24.88 3.82 6614.82 unknown e 31.38 1.75 56.04 24.54 3.92 8341.82 unknown f 31.58 1.69 49.29 25.29 3.70 4986.91 unknown g 32.09 1.54 35.06 25.36 3.68 4741.19 unknown h 32.18 1.52 33.03 25.45 3.65 4456.11 Using the calculated relative copy numbers above, relative copy number ratios were calculated. Using the formula stated above (A = 200 / (R- 1) where A = % T. aestivum and R = relative copy number ratio), % T. aestivum were calculated from the relative copy number ratios. D-genome Normaliser Calculated calculated relative calculated relative relative 'copy' no % 'copy' no 'copy' no ratio T. aestivum unknown a 39.25 5245.36 133.65 1.5 unknown b 39.25 5044.50 128.54 1.6 unknown c 66.15 9618.41 145.40 1.4 unknown d 50.28 6614.82 131.56 1.5 unknown e 56.04 8341.82 148.86 1.4 unknown f 49.29 4986.91 101.17 2.0 unknown g 35.06 4741.19 135.24 1.5 unknown h 33.03 4456.11 134.93 1.5

This technique can be applied to any material containing both genes of interest and the normalising genes and so may be applied to other situations. For example, mustard seed can be quantitatively tested to determine any contamination with rape seed or the seed of other brassicas. The invention is potentially applicable also to the establishment of absolute standards for use in the quantitative testing of any materials for genetically modified varieties, which will allow for the more accurate monitoring of genetically modified materials in foods and their raw materials. By means of the present invention, it is now possible to undertake the preparation of appropriate absolute standards and to make those standards available for commercial, industrial and legal testing and monitoring purposes.

Claims

CLA S:

1. The use of a standard in a method of nucleic acid sequence-based quantification on a mixture comprising a plurality of constituents, each constituent comprising a common nucleotide sequence, and a constituent of interest having a specific nucleotide sequence, characterised in that the standard is prepared from a single said constituent comprising the said common sequence and a constituent-specific sequence in a known or elucidatable fixed copy number ratio.

2. The use according to claim 1, wherein the standard is a single seed, part thereof or like specimen of a vegetable variety.

3. The use according to claim 1, wherein the standard is from a monoculture of an organism.

4. The use according to claim 1, wherein the standard is from tissue of an animal species.

5. The use according to any preceding claim, wherein the method is a DNA-based method of quantification.

6. The use according to any preceding claim, wherein the constituent-specific sequence is part of a naturally occurring sequence for the organism.

7. The use according to any preceding claim, wherein the constituent-specific sequence is a non-naturally occurring nucleotide sequence for the organism.

8. The use according to claim 5, wherein the DNA-based method is comprises PCR.

9. A method for preparing a standard for use in a method of nucleic acid sequence-based quantification on a mixture comprising a plurality of constituents, each constituent comprising a common nucleotide sequence and a constituent of interest having a specific nucleotide sequence, the method characterised by the step of preparing the standard from a single said constituent comprising the said common sequence and a constituent-specific sequence in a known or elucidatable, fixed copy number ratio.

10. A standard for use in a method of nucleic acid sequence-based quantification on a mixture comprising a plurality of constituents, each constituent comprising a common nucleotide sequence and a constituent of interest having a specific nucleotide sequence, characterised in that the standard comprises a single said constituent comprising the said common sequence and a constituent-specific sequence in a known or elucidatable fixed copy number ration.