Identification of transgenic dna sequences in plant material and products made of the same, oligonucleotide kit and bioarray therefor

FIELD: molecular biology, gene engineering.

SUBSTANCE: invention relates to method for detection of typical marker foreign DNA sequences, usable in plant modification, in transgenic plant materials and products made of the same. Claimed identification method includes carrying out of multiplex polymerase chain reaction on the base of DNA isolated from tested material followed by hybridization of fluorescent labeled reaction products on specialized bioarrays. Hybridization data are registered by control and program complex and identification of foreign genes and regulatory zones thereof in tested material is carried out, and obtained results are quantitatively interpreted.

EFFECT: method for large scale screening of plant materials and products obtained from the same.

5 cl, 5 dwg, 2 tbl, 4 ex

 

The technical field

The invention relates to the field of genetic engineering of plants, molecular biology, Biosafety, ficobiotehnologie and environmental protection, in particular to methods for detection and identification of typical insertion DNA used in genetic transformation of plants, genetically modified plant material and products based on it. The invention includes a specific set of oligonucleotide probes, the structure of the biochip, the method of sample preparation of plant DNA to the procedure of hybridization on the biochip, the methods of registration and interpretation of results.

The level of technology

For the detection of transgenic components of plant origin known method using antibodies to identify foreign proteins (Stave J.W. Food Control, 1999, vol.10, pp.367-374).

This way you can detect the presence of foreign proteins only known nature, with each possible transgenic protein you need to gain your type of antibody. Proteins themselves are normally unstable and during the processing of raw materials in the manufacture of food products can easily change the conformation and integrity, becoming consequently not available for detection with antibodies. The sensitivity of the method to detect foreign proteins on the basis of antibodies is not always sufficient for the reliable detection of small amounts of protein in transgenic products.

Another known method of detection of transgenic components of plant origin is the analysis of DNA using the method of polymerase chain reaction (NDP), in particular, to identify the composition of the DNA promoter 35S of cauliflower mosaic virus and nos terminator of Agrobacterium A.tumefaciens (Lipp M., Brodmann, P., Pietsch, K., Pauwels J., Anklam E. Journal of AOAC International, 1999, vol.82, No.4, pp.923-928).

The method is based on detecting in the test sample transgenic DNA is more sensitive and reliable than the method of analysis of proteins as DNA biopolymer more stable than protein. In addition, you will be able to identify not only the expressed portions of the genome, but also auxiliary, such as promoters and transcription terminators.

However, widely spread technique based on testing of a single promoter (35S) and one terminator (nos) using conventional PCR (Lipp M. et al., 1999) has a number of disadvantages and to date, ineffective, since a significant proportion (30-50%) of transgenic plants is created with the use of other promoters and terminators. In addition, the use of DNA fragments of phytopathogens as markers may lead to false-positive results if the original plants were infected with the respective viruses and/or bacteria. Detected using conventional PCR DNA fragments ioutline about 200 base pairs (Lipp M. et al., 1999), which means that in case of collapse of DNA, for example, by heat treatment of raw materials, to fragments smaller length this method of identification of transgenes is unsuitable. Also a serious drawback of traditional PCR is the visual nature of the evaluation results, which can lead to erroneous interpretations and is not possible to determine the quantitative relationships. It should be noted that the technique of PCR, using as DNA targets two determinants of transgenetic, unpromising as planned waiver application in the practice of genetic engineering of plant regulatory DNA fragments from pathogens.

Disclosure of the invention.

The present invention is to provide a novel method of identifying genetically modified plants and products on their basis, to eliminate the disadvantages of the known methods.

This problem is solved by a new method of detection of transgenic DNA sequences in plant material and products based on this material. In the claimed method proposed the use of multiplex PCR for the simultaneous amplification of fragments of genes and regulatory elements used in the transformation of the plant's DNA. The presence or absence of a set of transgenic sequences DN is determined using hybridization on selected oligonucleotide-probes, immobilized on made for this purpose, the biochips. The method also includes methods of recording and interpreting results.

The invention

The invention consists in a new approach for the detection of genetic determinants of transgenetic in plant material and products on its basis, using oligonucleotide biochip. The method is based on multiplex PCR using specific primers complementary to the sequences of the studied genes and regulatory elements, followed by hybridization of these fragments on the chip that contains the original set of differentiating oligonucleotides.

The use of these primers allows you to amplify fragments of small size - about 100 BP - transgenic DNA, if present in the genome of the test source. The resulting single-stranded fluorescently labeled DNA fragments are able to make specific hybridization interaction with oligonucleotides immobilized on the biochip. The famous order of the oligonucleotides on the chip gives the opportunity to establish what marker genes and regulatory elements are part of the transgenic DNA. Developed hybridization conditions provide a high degree of differentiation between those cells bi the chip, where hybridization occurred and those where specific hybrids have arisen. Design of primers and oligonucleotides provides absolute specificity of their interaction with potential transgenic DNA sequences. While immobilized oligonucleotide probe has a different sequence than the primers for the amplification, which excludes the possibility of hybridization in the cell any randomly amplified fluorescently labeled DNA. A visual check of the results of hybridization supplemented by a quantitative check on the basis of a specially adapted computer program that can accurately determine the degree of excess of the signal above the background level.

Schematic diagram of the detection of fragments of transgenic DNA

To the selection of the DNA sample add a few pairs of primers specific to the conservative regions of marker genes and regulatory regions in the DNA, usually used for transformation of plants. Carry out a multiplex PCR reaction in the selected mode. The accumulation of fluorescently labeled single-stranded PCR products is achieved due to the excess in each of the pairs of one of the primers containing 5'-end of the fluorescent label. The resulting PCR products hybridized on the chip with immobilized probes, complementary sequences, fragments of detective what's genes and/or regulatory elements.

Biochips represent an array of hydrogel microwells, fixed on a glass surface. In cells immobilized set of oligonucleotides homologous to fragments of transgenic DNA sequences. Each cell contains individual covalently immobilized oligonucleotide probe, and the immobilized oligonucleotides have a different sequence than the primers on the same genes. In addition, the biochip are cells with nonspecific oligonucleotides role of the negative control hybridization, and cells labeled with a fluorescent dye and intended for the correct orientation of the biochip. Position on the biochip all cells, both experienced and control, strictly deterministic. The surface of the biochip on which the microwells, closed plastikowymi housing hybridization chamber, which together with the glass forms a closed space, which serves to conduct hybridization. A camera body provided with a bent edge to remove the camera after carrying out hybridization and two holes to make the sample, which is sealed with masking tape.

The target DNA fragments perfect form stable hybridization duplexes only relevant (fully complementary) oligonucleotides. With all of Stalin the mi oligonucleotide-target DNA fragments can form only unstable imperfect duplexes. Differentiation of perfect and imperfect duplexes performed by comparing the fluorescence intensities of the cells, which formed duplexes. The intensity of the signal in the cell, which formed a perfect hybridization duplex, many times higher than in the cell, where he was able to form only an imperfect duplex. While the deterministic order of oligonucleotides in cells biochip provides an opportunity to establish what marker genes and regulatory elements are part of the transgenic DNA. The hybridization results can be interpreted unambiguously both visually and with the help of the program "Imageware", part of the hardware-software complex "Chipdetector-03" for analysis of fluorescence images obtained on the biochips.

Thus, one aspect of the invention is a set of specific oligonucleotides listed in table 1, are used as primers for PCR method for the identification of transgenic DNA sequences in plant material and products on the basis of such material.

Another aspect of the invention is a biochip, which is micrometric with cells, some of which contain immobilized oligonucleotide probe, other oligonucleotides to control the test procedure, and some f uorescent dyes for the correct orientation of the biochip at check results characterized in that the probes used oligonucleotides are shown in table 2, the complementary fragments of a typical marker and auxiliary DNA sequences used for genetic transformation of plants.

The next aspect of the invention is a method for the identification of transgenic DNA sequences in plant material and products on the basis of such material, including DNA analysis using polymerase chain reaction (PCR) and recording the results of the analysis, characterized in that: a) PCR performed on the DNA preparation in the form of a multiplex reaction with the production of fluorescently labeled product, using specific pairs of primers, one of which is fluorescently Machen and is present in excess relative to the second the unlabeled primer; b) PCR results analyzed by hybridization on the biochip, and C) the results of the analysis register with aparato-software complex, which shows the fluorescence of cells biochip on the computer screen, to quantify the fluorescent signal at any point in the biochip, to determine the ratio of the fluorescence signal to background in each cell biochip and accordingly the ratio of fluorescent signals in cells.

In the method according to the invention results recording hibri is Itachi on section 3(C) may be performed using a hardware-software complex "Chipdetector-03", which allows you to convert the results obtained in digital format and to make subsequent mathematical processing.

In addition, in the method according to the invention, if necessary, strengthen the hybridization signal of the PCR reaction according to claim 3(a) can be carried out in two stages, first as a symmetric multiplex PCR, and then as asymmetric multiplex PCR with the inclusion of fluorescently labeled primers.

Comparative analysis of the prototype allows us to conclude that the claimed method differs from the above method of identification of two regulatory elements transgenic DNA by conventional PCR on several parameters. First, the claimed method allows to significantly increase the number of detected targets and, at the same time, reduce the complexity due to the use of multiplex PCR. Secondly, the procedure gel electrophoresis of the prototype associated with the use of toxic substances and manual labor, replaced by the inventive method for hybridization on the biochip, which requires virtually no labor costs and staff involvement. Thirdly, the sizes amplificare fragments of the inventive method is substantially less than in the prototype that allows you to analyze significantly more degraded DNA. Fourthly, the application of the inventive method results with OSU special hardware-software complex "Chipdetector-03 allows you to set the intensity level of the fluorescent signal in each cell and interpreting the results of hybridization.

Brief description of drawings

Figure 1. Diagram of a biochip for the identification of transgenic plants.

Figure 2. The result of hybridization on the biochip. Detection of marker gene npt and gus, promoters nos and 35S in the sample DNA from potato cultivar Desiree.

Figure 3. The result of hybridization on the biochip. Detection of the marker gene npt, the nos promoter, the ocs terminator in the sample DNA from transgenic tobacco.

Figure 4. The result of hybridization on the biochip. Detection of the marker gene npt DNA sample of the food product.

Figure 5. The result of hybridization on the biochip DNA sample from nontransgenic plants.

The implementation of the invention

The claimed method is as follows. Immediately before use, prepare a mixture in microtubes for carrying out multiplex PCR, which comprises: the reaction buffer for PCR; the mixture deoxynucleotide-triphosphates at a concentration of 200 μm each; several pairs of primers in the amount of 5-10 pmoles for fluorescently labeled primers, and 0.5-2 pmole for unlabeled primers; 12.5 activity units of thermostable Taq DNA polymerase, and tested total DNA in the amount of 1-3 µg per sample. The composition of the primers are presented in table 1. Cones are placed in a programmable thermostat-thermal cycler and perform the PCR reaction as follows: denaturation at 95°30 (in the first cycle - 5 min), annealing at 60(±5)°C - 30 s, elongation at 72°S - 30 s (in the final cycle of 5 min), only about 35-45 cycles.

For carrying out hybridization of an aliquot of the sample obtained by PCR, mixed with hybridization buffer with bringing components to last the following concentrations: GuSCH - 1 M HEPES, pH 7.5-50 mm, EDTA 5 mm. The resulting mixture was placed on a biochip, sealed hybridization chamber and carry out hybridization at 37°C for 14-18 hours. After hybridization biochip rinsed three times with distilled water at 25°and dried.

Registration hybridization pattern is carried out, using a hardware-software complex "Chipdetector program Imageware.

Manufacturer oligonucleotide biochip for the detection of marker genes and regulatory regions in the DNA used for transformation of plants

Oligonucleotides are synthesized on an automated synthesizer (type 394 DNA/RNA synthesizer. Applied Biosystems, USA) and contain a spacer with a free amino group of 3'-Amino-Modifier C7 CPG 500 (Glen Research, USA) for subsequent immobilization in gel or 5'-Amino-Modifier C6 (Glen Research, USA) for the introduction of fluorescent labels. The introduction of fluorescent labels su-5 ("Biochip-MPI", Russia) is carried out in accordance with the manufacturer's recommendations.

Gel cell biochip is applied onto a glass substrate in the form of droplets mA the maximum accuracy and reproducibility using robots (Rubina A.Y. et al. Anal Biochem., 2004, vol.325(1), pp.92-106). Each generated cell is a hemisphere with a diameter of 200-250 μm and approximately 300 μm and contains immobilized oligonucleotide probes, complementary to the sequence of a gene or regulatory element (a promoter or terminator)used in the genetic modification of plants.

The structure of the biochip

Biochip contains five of immobilized oligonucleotide probes, the list of which is presented in table 2, three marker points for correct positioning (image capture)performed by 'Imageware', and the three cells containing non-specific to the studied genomes oligonucleotides role of the negative control. Location immobilized on the microchip oligonucleotides shown in figure 1.

Sequence immobilized oligonucleotides are given in table 2.

Registration hybridization pattern is carried out, using a hardware-software complex "Chipdetector-03".

The interpretation of the results of hybridization is carried out by comparing the intensity of the fluorescent signal in cells 35S_I, gus_I, nos_I, npt_I, ocs_I with the intensity of the signal in NC cells (figure 1)negative control. If the ratio of at least one of these signals exceed the threshold value determined for each of analizirue who's cell and asked in the "Imageware", then follows the conclusion that the investigated sample is transgenic, i.e. contains foreign DNA.

Examples

Example 1.

Analysis of the presence of foreign sequences in DNA from transgenic potato.

DNA from transformed potato cultivar Desiree was isolated by the standard method and used in the above-described test procedure. The primers used were and immobilized oligonucleotides for marker gene npt II and gus, for a promoter 35S of cauliflower mosaic virus, the nos promoter and ocs terminator of Agrobacterium A.tumefaciens.

Preparation of samples for hybridization on the microarray method for multiplex PCR

In 27 μl of PCR mixture was made 3 μl of DNA solution extracted from the studied sample. The composition of the PCR mixture:

1×PCR buffer: 50 mm KCl, 10 mm Tris-HCl (pH 9.0 at 25°C), 0.1% Triton® X-100 (sileks, Russia)

2 mm MgCl2(Sileks, Russia)

200 μm of each dNTP (sileks, Russia)

12.5 units of thermostable Taq DNA polymerase (sileks, Russia)

1 µl of a mixture of primers (table 1).

Amplification was performed in a programmable thermal cycler "Terzic" (DNA-Technology, Russia) with the following regime: 95°C - 30 s, 62°C - 30 s, 72°C - 30; 43 loop (in the first cycle, the denaturation time was increased to 5 min, in the last - time extension of up to 5 min).

Figure 2 presents a picture of hybridization on the biochip is fluorescently labeled PCR products. It is seen that four cells emit a strong fluorescent signal that is many times greater than the background fluorescence of the cells of the negative control NC. Fluorescent cells contain sequences gene npt II and gus and 35S promoter and nos. The cell corresponding to the ocs terminator, kept the background fluorescence. The result of this test can be unambiguously interpreted as positive, i.e. identifying the transgenic nature of plant sample. This testing establishes that in the plant genome introduced marker genes npt II and gus and 35S promoter and nos.

Example 2.

Analysis of the presence of foreign sequences in DNA from transgenic tobacco. The test was carried out similarly as described in example 1.

Figure 3 presents a picture of hybridization on the biochip fluorescently labeled PCR products.

It is seen that three cells emit a strong fluorescent signal that is many times greater than the background fluorescence of the cells of the negative control NC. Fluorescent cells contain a gene npt II, ocs terminator, a promoter nos. Cells containing oligonucleotides, complementary to the fragments of ocs and 35S, kept the background fluorescence.

The result of this test can be unambiguously interpreted as positive, i.e. detecting transgenic nature given the CSOs plant sample. This testing establishes that in the plant genome embedded npt II gene, and the nos promoter, the ocs terminator.

Example 3.

Analysis of the presence of foreign DNA sequences from food (pate "Popular", Cherkizovsky plant). The test was carried out similarly as described in example 1. Picture hybridization on the biochip fluorescently labeled PCR products are presented in figure 4.

Fluorescent signal is observed only in the cell containing the oligonucleotide complementary to the sequence of a fragment of the gene npt II. In all other analyzed cells biochip intensity of the signal is close to the value of the intensity in the cells of the negative control. Therefore, the analyzed food product contains genetically modified plant component that is installed on the presence of the npt II gene.

Example 4.

Analysis of the presence of foreign DNA sequences nontransgenic plants. The test was carried out similarly as described in example 1. Picture hybridization on the biochip fluorescently labeled PCR products are presented in figure 5.

The results show that none of the experimental cells does not give a fluorescent signal significantly greater than the control signal cells. This indicates neresenog nature of the tested DNA.

The main advantage of PR is degenova method is the simultaneous analysis of presence in the tested DNA immediately a large number of different genes and their regulatory regions, usually used to obtain transgenic plants. This makes it possible not only to reliably discriminate against transgenic and necroshine DNA samples, but also to clearly identify what specific marker genes are present in the transgenic material. However, a greater number of detected sequences does not make the procedure more difficult to perform, because stage PCR amplification of DNA fragments, and the stage of hybridization on the microarray are performed in one step regardless of the number of test sequences. A large number of test DNA sequences is unlikely to produce false negative results, since the probability to nd at least one fragment of foreign DNA is coming here to 100%. It is also important that the transgenic plants have, as a rule, several different built-in genome sequences. Therefore, in the presence of transgenic material in the original sample fluorescent signal will often be observed in several cells with probes specific for different targets, each of which serves as an independent confirmation of the positive test result, which reduces the likelihood of false-positive results.

The examples illustrate the advantages of the present invention for identification of transgenic components only of the part, because these benefits are not limited to the above examples, and due to the possibility to Supplement the composition of the biochip as far as the genetic engineering of new regulatory regions in the DNA and the marker gene to create transgenic plants.

To achieve the high reliability of the proposed method is additionally implemented the following measures. First, immobilized probes correspond to the inner part amplificare specific fragments of DNA targets. This eliminates the possibility of non-specific hybridization of any randomly amplified DNA. Secondly, the structure of the biochip entered the cell, performing the role of the negative control. Thirdly, the length amplificare of DNA fragments of approximately 100 BP, which gives the opportunity to identify transgenic sequences even in severely degraded to the lengths of 150-200 concentration DNA samples. In addition, in the process of testing each series of samples provided by the use of positive and negative controls compared DNA samples isolated from characterized transgenic and nedrencheskogo plants.

High sensitivity and specificity of the method is due to the fact that it combines the advantages of two of the most sensitive and specific methods of genome analysis: polymerase chain reaction (PCR) and the special is practical hybridization. PCR increases the concentration of desired DNA sequences in a million or more times, which is sufficient for reliable and quantitative detection of the fluorescent signal. Hence, for reliable detection of transgenes in the original DNA is sufficient only 105-106the same transgenic sequences, which corresponds to approximately 1-10 FG, ie 10-15-10-14g their DNA. This sensitivity corresponds to one of the most high for the currently known methods for the detection of transgenes.

The proposed method is high-tech and does not require significant labor costs. Components for PCR can be prepared in advance in the same mixture, and the PCR reaction is no more than 1.5 hours. Hybridization on the biochip is held without the participation of staff and can also be carried out outside working hours, i.e. during the night. Washing of the chip after hybridization and fluorescence analysis of cells on a hardware-software complex "Chipdetector-03" require minimum labour costs and can be completed within minutes. The shelf life of biochips with prehybridization fluorescently labeled material is several years, with re-registration of fluorescence can be carried out at any time. Complex Chipdetector-03" allows you to save in computer memory as images, so cifrovye the intensities of fluorescent signals.

Thus, the convenience and speed of testing, as well as the objectivity of the analysis allow us to test a large number of DNA samples in parallel in strictly identical conditions, without requiring any of the stages of highly qualified personnel. While retaining indefinitely the possibility of re-analysis of the biochip after hybridization.

The proposed method does not pose health risks to personnel and to the environment, it is not associated with the use of toxic or radioactive compounds. Due to the fact that in the process of testing shall be used only microvolumes of liquid media, there is no problem with recycling or waste disposal.

Table 2.

The list of oligonucleotides immobilized on the biochip
SEQ ID

No:
The name of the oligonucleotideDetected targetThe sequence of the oligonucleotide [5'-NH2-(B)n]
1135S_I35S-promoter5' GCC ATC ATT GCG ATA AAG G
12gus_Igene gus5' CTG GTA TCA GCG CGA A
13nos_Ithe nos promoter5' GCT AAG CAC ATA CGT CAG AA
14npt_Igene nptII5' GGC AGC GCG GCT ATC
15ocs_Ithe ocs terminator5' TTC TGT TGT GCA CGT TGT A

1. The method of identification of transgenic DNA sequences in plant material and products on the basis of such material, including DNA analysis using polymerase chain reaction (PCR) and recording the results of the analysis, wherein a) PCR performed on the DNA preparation in the form of a multiplex reaction with the production of fluorescently labeled product, using specific pairs of primers, one of which is fluorescently Machen and is present in excess relative to the second the unlabeled primer; b) PCR results analyzed by hybridization on the biochip, and C) the results of the analysis register using a hardware-software complex allows to display fluorescence cell biochip on the computer screen, to quantify the fluorescent signal at any point in the biochip, to determine the ratio of the fluorescence signal to background in each cell biochip and accordingly the ratio of fluorescent signals in cells.

2. The method according to claim 1, characterized in that the registration of the hybridization results on section 3(C) is performed using a hardware-software complex "Chipdetector-03", that call is employed to convert the results obtained in digital format and to make subsequent mathematical processing.

3. The method according to claim 1, characterized in that to enhance the hybridization signal of the PCR reaction according to claim 3(a) is carried out in two stages, first as a symmetric multiplex PCR, and then as asymmetric multiplex PCR with the inclusion of a fluorescently labelled primers.

4. The set of specific oligonucleotides listed in table 1, are used as primers for PCR method for the identification of transgenic DNA sequences in plant material and products on the basis of such material, characterized in claim 1.

5. Biochip, which is micrometric with cells, some of which contain immobilized oligonucleotide probe, other oligonucleotides to control the test procedure, and some fluorescent dyes for the correct orientation of the biochip during the registration results, characterized in that the probes used oligonucleotides are shown in table 2, the complementary fragments of a typical marker and auxiliary DNA sequences identified by the method described in claim 1.



 

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