RU2270254C2 - Identification of transgenic dna sequences in plant material and products made of the same, oligonucleotide kit and bioarray therefor - Google Patents

Abstract

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

Description

Technical field

The invention relates to the field of plant genetic engineering, molecular biology, biosafety, phytobiotechnology and environmental protection, in particular to methods for identifying and identifying typical DNA insertions used in the genetic transformation of plants in genetically modified plant material and products based on it. The invention includes a specific set of oligonucleotide probes, a biochip structure, a method for preparing a plant DNA sample for hybridization on a biochip, methods for recording and interpreting the results.

State of the art

To detect transgenic components of plant origin, a method is known for using antibodies to identify foreign proteins (Stave J.W. Food Control, 1999, vol. 10, pp. 367-374).

In this way, it is possible to detect the presence of foreign proteins of only a known nature, and each type of transgenic protein needs to develop its own type of antibody. Proteins themselves are usually unstable and, during the processing of raw materials in the manufacture of food products, can easily change conformation and integrity, thereby becoming inaccessible to detection with antibodies. The sensitivity of the method for detecting foreign antibodies-based proteins is not always sufficient for reliable detection of small amounts of protein in transgenic products.

Another known method for detecting transgenic components of plant origin is DNA analysis using the method of polymerase chain reaction (NDP), in particular, to identify the 35S promoter of the cauliflower mosaic virus and the nos terminator of the 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 based on the detection of transgenic DNA in the test samples is more sensitive and reliable than the protein analysis method, because DNA as a biopolymer is more stable than protein. In addition, it becomes possible to identify not only expressed genome regions, but also auxiliary ones, such as promoters and transcription terminators.

However, the widely used technique based on testing one promoter (35S) and one terminator (nos) using traditional PCR (Lipp M. et al., 1999) has a number of disadvantages and is currently ineffective, because a significant proportion (up to 30-50%) of transgenic plants is created using other promoters and terminators. In addition, the use of DNA fragments of phytopathogens as markers can lead to false-positive results if the original plants were infected with the corresponding viruses and / or bacteria. DNA fragments detected by conventional PCR have a length of about 200 base pairs (Lipp M. et al., 1999), which means that in the case of DNA decomposition, for example, by heat treatment of the feedstock, this method of transgenic identification turns out to be shorter fragments unsuitable. Another serious drawback of the traditional PCR method is the visual nature of the evaluation of the results, which can lead to their false interpretation and does not allow to determine the quantitative ratio. It should be noted that the described PCR method using two determinants of transgenicity as DNA targets is unpromising, because It is planned to abandon the use of regulatory DNA fragments from pathogenic microorganisms in the practice of plant genetic engineering.

Disclosure of the invention.

The objective of the present invention is to provide a new method for identifying genetically modified plants and products based on them, to eliminate the disadvantages of known methods.

This problem is being solved by a new method for detecting transgenic DNA sequences in plant material and products based on this material. In the claimed method, the use of multiplex PCR for the simultaneous amplification of gene fragments and regulatory elements used in the transformation of plant DNA is proposed. The presence or absence of a set of transgenic DNA sequences is determined by hybridization on specially selected probe oligonucleotides immobilized on biochips made for this purpose. The method also includes methods for recording and interpreting analysis results.

SUMMARY OF THE INVENTION

The essence of the invention is a new approach for the detection of genetic determinants of transgenicity in plant material and products based on it, using an 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 a biochip containing an original set of differentiating oligonucleotides.

The use of these primers allows amplification of small fragments of about 100 bp. - transgenic DNA, if any, is present in the genome of the test source. The resulting single-stranded fluorescently labeled DNA fragments are able to enter into specific hybridization interactions with oligonucleotides immobilized on a biochip. The known arrangement of oligonucleotides on the biochip makes it possible to determine which marker genes and regulatory elements are part of transgenic DNA. The developed hybridization conditions provide a high degree of differentiation between those cells of the biochip where hybridization has occurred and those where specific hybrids have not arisen. The design of primers and oligonucleotides provides the absolute specificity of their interaction with the desired transgenic DNA sequences. In this case, the immobilized oligonucleotide has a different sequence than the amplification primers, which excludes the possibility of hybridization in the cell of any randomly amplified fluorescently labeled DNA. Visual registration of the results of hybridization is supplemented by quantitative registration based on a specially adapted computer program that allows you to accurately determine the degree to which the signal exceeds the background level.

The concept of detecting fragments of transgenic DNA

Several pairs of primers specific to the conserved regions of marker genes and regulatory regions of DNA commonly used in plant transformation are added to the DNA extracted from the sample. Conduct a multiplex PCR reaction in the selected mode. The accumulation of fluorescently labeled single-chain PCR products is achieved due to the excess in each of the pairs of one of the primers containing a fluorescent label at the 5'-end. The resulting PCR products are hybridized on a biochip with immobilized probes complementary to the sequences of fragments of the detected genes and / or regulatory elements.

Biochips are an array of hydrogel microcells mounted on a glass surface. A set of oligonucleotides homologous to fragments of transgenic DNA sequences is immobilized in the cells. Each cell contains an individual covalently immobilized oligonucleotide, and the immobilized oligonucleotides have a different sequence than the primers for the same genes. In addition, cells with non-specific oligonucleotides are located on the biochip, which play the role of a negative control of hybridization, as well as cells marked with a fluorescent dye and intended for the correct orientation of the biochip. The position on the biochip of all cells, both experimental and control, is strictly determined. The surface of the biochip, on which the microcells are located, is closed by the plastic casing of the hybridization chamber, which together with the glass forms an enclosed space serving for hybridization. The camera body is equipped with a bent edge to remove the camera after hybridization and two holes for the introduction of the sample, which are sealed with adhesive tape.

The studied DNA fragments form perfect stable hybridization duplexes only with the corresponding (fully complementary) oligonucleotides. With all other oligonucleotides, the studied DNA fragments can form only unstable imperfect duplexes. The differentiation of perfect and imperfect duplexes is performed by comparing the fluorescence intensities of the cells in which the duplexes formed. The signal intensity in the cell in which the perfect hybridization duplex was formed is many times higher than in the cell where only imperfect duplex could be formed. In this case, the determinate arrangement of oligonucleotides in the cells of the biochip makes it possible to establish which marker genes and regulatory elements are part of transgenic DNA. Hybridization results can be interpreted uniquely both visually and using the Imageware program, which is part of the Chipdetector-03 hardware and software complex for analyzing fluorescence images obtained on biochips.

Thus, one aspect of the invention is a set of specific oligonucleotides shown in Table 1, used as primers for PCR in a method for identifying transgenic DNA sequences in plant material and products based on such material.

Another aspect of the invention is a biochip, which is a microarray with cells, some of which contain immobilized probe oligonucleotides, others are oligonucleotides for monitoring the test procedure, and others are fluorescent dyes for the correct orientation of the biochip when recording results, characterized in that as probes used oligonucleotides shown in table 2, complementary to fragments of typical marker and auxiliary DNA sequences used in genetic plant transformation.

Another aspect of the invention is a method for identifying transgenic DNA sequences in plant material and products based on such material, including DNA analysis by polymerase chain reaction (PCR) and recording the results of the analysis, characterized in that: a) PCR is carried out on the DNA preparation in the form of multiplex reactions to obtain a fluorescently labeled product, using specific pairs of primers, one of which is fluorescently labeled and is present in excess relative to the second unlabeled mu primer; b) the PCR results are analyzed using hybridization on a biochip and c) the analysis results are recorded using an hardware-software complex that displays the fluorescence of the biochip cells on a computer screen, quantifies the fluorescence signal at any point of the biochip, determines the ratio of the fluorescent signal to the background in each cell biochip and, accordingly, the ratio of fluorescent signals in the cells.

In the method according to the invention, the registration of the results of hybridization according to claim 3 (c) can be performed using the Chipdetector-03 hardware-software complex, which allows you to convert the obtained results in digital format and perform subsequent mathematical processing.

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

Comparative analysis with the prototype allows us to conclude that the claimed method differs from the above method for identifying two regulatory elements of transgenic DNA using traditional PCR in several ways. Firstly, the claimed method can significantly increase the number of detected targets and, at the same time, reduce the complexity due to the use of multiplex PCR. Secondly, the prototype gel electrophoresis procedure associated with the use of toxic substances and manual labor is replaced in the claimed method with a biochip hybridization that practically does not require labor costs and personnel participation. Thirdly, the size of the amplified fragments in the claimed method is significantly smaller than in the prototype, which allows you to analyze a significantly more degraded DNA. Fourth, the application in the claimed method of recording results using a special hardware-software complex "Chipdetector-03" allows you to set the intensity level of the fluorescent signal in each cell and uniquely interpret the results of hybridization.

Brief Description of the Drawings

Figure 1. Biochip scheme for identification of transgenic plant objects.

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

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

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

Figure 5. The result of hybridization on a biochip of a DNA sample from an untransformed plant.

The implementation of the invention

The claimed method is as follows. Immediately before the test, a mixture is prepared in microtubes for conducting multiplex PCR, which includes: a reaction buffer for PCR; a mixture of deoxynucleotide triphosphates in a concentration of 200 μm each; several pairs of primers in an amount of 5-10 pmoles for fluorescently labeled primers and 0.5-2 pmoles for unlabeled primers; 12.5 activity units of thermostable Taq DNA polymerase, as well as the test total DNA in the amount of 1-3 μg per sample. The composition of the primers is presented in table 1. The microtube is placed in a programmable thermostat-thermocycler and the PCR reaction is carried out in the following mode: denaturation at 95 ° C for 30 s (in the first cycle for 5 min), annealing at 60 (± 5) ° C for 30 s, elongation at 72 ° С - 30 s (in the last cycle - 5 min), a total of about 35-45 cycles.

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

Registration of the hybridization pattern is carried out using the hardware-software complex Chipdetector with Imageware.

Production of an oligonucleotide biochip for detecting marker genes and regulatory regions of DNA used in plant transformation

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

The gel cells of the biochip are applied on a glass substrate in the form of drops with maximum accuracy and reproducibility using robots (Rubina A.Y. et al. Anal Biochem., 2004, vol. 325 (1), pp. 92-106). Each formed cell is a hemisphere with a diameter of 200-250 microns and a period of about 300 microns and contains an immobilized oligonucleotide probe complementary to the sequence of a gene or regulatory element (promoter or terminator) used in the genetic modification of plants.

Biochip structure

The biochip contains five immobilized probe oligonucleotides, the list of which is presented in Table 2, three marker points for the correct positioning (image capture) performed by the Imageware program, and three cells containing oligonucleotides that are non-specific to the genome under study and that act as a negative control. The location of the oligonucleotides immobilized on the microchip is shown in FIG.

The sequences of immobilized oligonucleotides are shown in Table 2.

The registration of the hybridization picture is carried out using the hardware-software complex Chipdetector-03.

Interpretation of the hybridization results 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 signal intensity in NC cells (Fig. 1), which serve as a negative control. If the ratio of at least one of these signals exceeded the threshold value determined for each of the analyzed cells and specified in the Imageware program, then the conclusion follows that the test sample is transgenic, i.e. contains foreign DNA.

Examples

Example 1

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

DNA from transformed Desiree potatoes was isolated by the standard method and used in the test procedure described above. Primers and immobilized oligonucleotides were used for the npt II and gus marker genes, as well as for the 35S promoter of cauliflower mosaic virus, the nos promoter, and the ocs terminator of the agrobacterium A.tumefaciens.

Sample preparation for hybridization on a microchip by multiplex PCR

In 27 μl of the PCR mixture, 3 μl of the DNA solution isolated from the test sample was added. 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 MgCl ₂ (Sileks, Russia)

200 μm of each dNTP (Sileks, Russia)

12.5 units thermostable Taq DNA polymerase (Sileks, Russia)

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

Amplification was carried out on a Tertsik programmable thermal cycler (DNA-Technology, Russia) with the following mode: 95 ° C for 30 s, 62 ° C for 30 s, 72 ° C for 30 s; 43 cycles (in the first cycle, the denaturation time was increased to 5 minutes, in the last - completion time to 5 minutes).

Figure 2 presents the picture of hybridization on a biochip of fluorescently labeled PCR products. It can be seen that four cells give a strong fluorescent signal, many times higher than the background fluorescence of the cells of the negative control NC. Fluorescent cells contain sequences of npt II and gus genes, as well as 35S and nos promoters. The cell corresponding to the ocs terminator retained background fluorescence. The result of this test can be unambiguously interpreted as positive, i.e. revealing the transgenic nature of this plant sample. The same testing establishes that npt II and gus marker genes, as well as 35S and nos promoters, are introduced into the plant genome.

Example 2

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

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

It can be seen that three cells give a strong fluorescent signal, many times higher than the background fluorescence of the cells of the negative control NC. Fluorescent cells contain sequences of the npt II gene, the ocs terminator, and the nos promoter. Cells containing oligonucleotides complementary to ocs and 35S fragments retained background fluorescence.

The result of this test can be unambiguously interpreted as positive, i.e. revealing the transgenic nature of this plant sample. The same testing establishes that the npt II gene, as well as the nos promoter, ocs terminator, are introduced into the plant genome.

Example 3

Analysis of the presence of alien DNA sequences from a food product (Paste Popular, Cherkizovsky Plant). The test was carried out similarly to that described in example 1. The pattern of hybridization on a biochip of fluorescently labeled PCR products is presented in figure 4.

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

Example 4

Analysis of the presence of foreign DNA sequences of an untransformed plant. The test was carried out similarly to that described in example 1. The pattern of hybridization on a biochip of fluorescently labeled PCR products is presented in figure 5.

The results show that none of the experimental cells gives a fluorescent signal that significantly exceeds the signal of the control cells. This indicates the non-transgenic nature of the tested DNA.

The main advantage of the proposed method is the simultaneous analysis of the presence in the test DNA of a large number of different genes and their regulatory regions, usually used to obtain transgenic plants. This allows not only to reliably discriminate transgenic and non-transgenic DNA samples, but also to uniquely determine which specific marker genes are present in the transgenic material. Moreover, a large number of detected sequences does not make the procedure more difficult to execute, because the stage of PCR amplification of DNA fragments and the stage of hybridization on a microchip are performed in one step, regardless of the number of test sequences. A large number of DNA sequences tested makes false negative results unlikely, as the probability of detecting at least one fragment of foreign DNA is approaching 100% here. It is also important that transgenic plants usually have several different sequences built into the genome. Therefore, in the case of the presence of transgenic material in the initial sample, the fluorescent signal will most often be observed in several cells with probes specific to different targets, each of which serves as an independent confirmation of a positive test result, which reduces the likelihood of false positive results.

The above examples illustrate the advantages of the present invention for the identification of transgenic components only partially, because these advantages are not limited to the given examples and are due to the ability to constantly supplement the biochip composition as genetic engineering assumes new regulatory DNA regions and marker genes to create transgenic plants.

To achieve high reliability of the proposed method, the following measures are additionally implemented. First, immobilized probes correspond to the inside of amplified specific fragments of target DNA. This eliminates the possibility of nonspecific hybridization of any randomly amplified DNA. Secondly, cells performing the role of negative control were introduced into the structure of the biochip itself. Thirdly, the lengths of amplified DNA fragments are about 100 bp, which makes it possible to detect transgenic sequences even in highly degraded to lengths of 150-200 bp DNA samples. In addition, in the process of testing each series of samples, the use of positive and negative control comparisons is provided - DNA samples isolated from characterized transgenic and non-transgenic plants.

High sensitivity and specificity of the method are due to the fact that it combines the advantages of the two most sensitive and specific methods of genome analysis: polymerase chain reaction (PCR) and specific hybridization. PCR increases the concentration of the desired DNA sequences by a million or more times, which is quite sufficient for reliable and quantitative detection of the fluorescent signal. Based on this, for the reliable detection of transgenes in the initial DNA, it is sufficient to have only 10 ⁵ -10 ⁶ identical transgenic sequences, which corresponds to about 1-10 fg, i.e. 10 ^-15 -10 ^-14 g of their DNA. Such sensitivity corresponds to one of the highest for currently known methods for detecting transgenes.

The proposed method is high-tech and does not require significant labor costs. Components for PCR can be prepared in advance in one mixture, and the PCR reaction itself takes no more than 1.5 hours. Hybridization on a biochip takes place without the participation of personnel and can be carried out after hours, i.e. during the night. Washing the chip after hybridization and analysis of cell fluorescence on the Chipdetector-03 hardware-software complex require a minimum of labor costs and can be carried out within a minute. The shelf life of biochips with a hybridized fluorescently labeled material is several years, and fluorescence can be re-registered at any time. The Chipdetector-03 complex allows you to save both images and digital values of fluorescence signal intensities in the computer memory.

Thus, the convenience and speed of testing, as well as the objectivity of the analysis, allow testing of a large number of DNA samples in parallel under strictly identical conditions, without requiring highly qualified personnel at any stage. At the same time, the possibility of repeated analysis of the biochip after hybridization is retained for an unlimited time.

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

Table 2.
List of oligonucleotides immobilized on a biochip SEQ ID
No: The name of the oligonucleotide Detectable target The sequence of the oligonucleotide [5'-NH ₂ - (B) _n ] eleven 35S_I 35S promoter 5 'GCC ATC ATT GCG ATA AAG G 12 gus_I gus gene 5 'CTG GTA TCA GCG CGA A 13 nos_I nos promoter 5 'GCT AAG CAC ATA CGT CAG AA fourteen npt_I nptII gene 5 'GGC AGC GCG GCT ATC fifteen ocs_I terminator ocs 5 'TTC TGT TGT GCA CGT TGT A

Claims (5)

1. A method for identifying transgenic DNA sequences in plant material and products based on such material, including DNA analysis by polymerase chain reaction (PCR) and recording the results of the analysis, characterized in that a) PCR is performed on the DNA preparation in the form of a multiplex reaction to obtain fluorescently labeled product, using specific pairs of primers, one of which is fluorescently labeled and is present in excess relative to the second unlabeled primer; b) the PCR results are analyzed using hybridization on a biochip and c) the analysis results are recorded using a hardware-software complex that displays the fluorescence of the biochip cells on a computer screen, quantifies the fluorescence signal at any point of the biochip, determines the ratio of the fluorescent signal to the background in each cell biochip and, accordingly, the ratio of fluorescent signals in the cells. 2. The method according to claim 1, characterized in that the registration of the results of hybridization according to claim 3 (c) is carried out using the hardware-software complex Chipdetector-03, which allows you to convert the results in digital format and perform subsequent mathematical processing. 3. The method according to claim 1, characterized in that to enhance the hybridization signal, the PCR reaction according to claim 3 (a) is carried out in two stages, first as a symmetric multiplex PCR, and then as an asymmetric multiplex PCR with the inclusion of fluorescently labeled primers. 4. A set of specific oligonucleotides shown in table 1, used as primers for PCR in the method of identification of transgenic DNA sequences in plant material and products based on such material described in paragraph 1. 5. A biochip, which is a microarray with cells, some of which contain immobilized probe oligonucleotides, others contain oligonucleotides to control the testing procedure, and others contain fluorescent dyes to correctly orient the biochip when recording results, characterized in that oligonucleotides are used as probes shown in table 2, complementary to fragments of typical marker and auxiliary DNA sequences identified by the method described in claim 1.

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