RU2386698C1 - Method of identifying transgenic dna sequences in plant material and products based on plant material - Google Patents

Abstract

FIELD: chemistry; biochemistry.

SUBSTANCE: DNA of plant material or products based on plant material is analysed through PCR using specific primers and fluorescent-labeled probes, complementary DNA sequences. Analysis results are recorded using a fluorescent PCR detector, where amplification products are detected in closed amplification vials. This prevents the need to move the amplification products into biochip cells after PCR, completely avoids contamination of reagents and equipment with PCR products and avoids obtaining erroneous analysis results. Vials which are prepared beforehand with a sealed paraffin mixture for amplification are stored for a long time at room temperature, which enables their use as part of ready diagnosis sets. If necessary, additional sets of primers and fluorescent-labeled probes can be used for analysing a wide range of transgenic DNA sequences used in engineering transgenic plants.

EFFECT: possibility of analysing a wide range of transgenic DNA sequences used in engineering transgenic plants.

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Description

Application area

The invention relates to the field of plant genetic engineering, molecular biology, food biosafety, phytobiotechnology and environmental protection. The invention can be used to identify and identify typical DNA insertions used in the genetic transformation of plants in genetically modified plant material and products based on it.

State of the art

To detect transgenic components of plant origin, a DNA analysis method using the polymerase chain reaction (PCR) method using specific primers complementary to transgenic DNA sequences is known (Lipp M., Brodmann P., Pietsch K., Pauwels J., Anklam E. Journal of AOAC International, 1999, vol. 82, No. 4, pp. 923-928).

The closest method is a method for identifying transgenic DNA sequences in plant material and products based on such material, involving the use of a biochip to analyze PCR results. (RF patent 2270254, Mirzabekov A.D., Gryadunov D.A., Mikhailovich V.M., Zasedatelev A.S., Romanov G.A., Kuznetsov V.V., Tsydenedambaev V.D., Mitrokhin I. A., Krylova E.M., publ. 02.20.2006).

The main disadvantage of this method is the need to transfer the amplification after PCR to the biochip cells, which creates the prerequisite for contamination of reagents and laboratory equipment with PCR products and can lead to false analysis results.

In addition, pre-prepared tubes with a mixture for amplification cannot be used.

A task

The present invention is the creation of a new method for identifying transgenic DNA sequences in plant material and products based on this material to eliminate the above disadvantages.

Disclosure of invention

This problem is solved by a new method for identifying transgenic DNA sequences in plant material and products based on this material, including DNA analysis by PCR using a set of oligonucleotides - specific primers and fluorescently labeled probes complementary to these sequences, and detection of PCR results, and PCR amplification is carried out using a set of oligonucleotides (table 1), with the detection of amplification results in closed amplification tubes.

SUMMARY OF THE INVENTION

The essence of the invention lies in a new approach for the detection of transgenic DNA sequences in plant material and products based on this material. The inventive method is based on PCR using specific primers and fluorescently labeled probes complementary to transgenic DNA sequences (table 1), followed by detection of amplification products in closed amplification tubes using a fluorescence PCR detector. A key element of the proposed method is the use of fluorescently labeled probes, which are oligonucleotides labeled with fluorophore and fluorescence quencher molecules. Probes are added to the reaction mixture along with primers and other components of the reaction. During PCR, the fluorescently labeled probe hybridizes to specific amplicons and is destroyed by Taq polymerase, as a result of which the fluorophore is free from the quencher. Thus, the level of fluorescence in amplification tubes increases in proportion to the number of specific PCR products formed.

Comparative analysis with the prototype allows us to conclude that the novelty and inventive step of the claimed invention lies in the fact that PCR using a set of oligonucleotides (table 1) and detection of PCR results in closed amplification tubes completely eliminates contamination of reagents and equipment with PCR products and prevents the appearance of false analysis results.

The implementation of the invention

The concept of detecting fragments of transgenic DNA

DNA extracted from the sample is added to the amplification mixture containing primers and fluorescently labeled probes specific for transgenic sequences, then the amplification of the DNA is carried out using an amplifier, and the amplification results are detected in closed amplification tubes after completion of PCR using a fluorescent PCR detector .

Concrete example

Analysis of the presence of foreign DNA sequences in plant material (GM soybean line 40-3-2).

DNA from beans of the GM soybean line 40-3-2 is isolated by the standard method. An equal amount of DNA (5 μl) isolated from each test sample was analyzed using four sets of DNA amplification reagents, differing in sets of primers and fluorescently labeled probes specific for the 35S promoter, nos promoter, gus gene, and nptII gene, respectively. The sequences of primers and oligonucleotide probes are shown in table 1. In addition to the DNA of the studied samples, the positive and negative control samples are analyzed using the indicated sets of reagents.

These sets of reagents for amplification of DNA in addition to specific primers (14 pM each) and fluorescently labeled probes (7 pM) contain the following common components: 6 mM of each dNTP; 84 mM TRIS-HCl (pH 8.6); 21 mM (NH ₄ ) ₂ SO ₄ ; 3.1 mmol - MgCl ₂ as well as 5 units of Taq polymerase. The total volume of the reaction mixture is 35 μl. For each set of reagents, background tubes are prepared, characterized by the absence of Taq polymerase, into which a negative control sample is added and thermocycled together with the remaining tubes.

Amplification is carried out using the Tercik amplifier manufactured by NPF DNA-Technology CJSC (Moscow) at the following temperature conditions: initial heating at 94 ° С for 1 min 30 sec, denaturation at 94 ° С - 20 sec, annealing at 64 ° C - 5 sec, elongation at 67 ° C - 5 sec (5 cycles), then denaturation at 94 ° C - 1 sec, annealing at 64 ° C - 5 sec, elongation at 67 ° C - 5 sec (40 cycles).

Detection of amplification products in closed tubes is carried out at the end of PCR using a fluorescence PCR detector "Gene". Interpretation of the results is carried out automatically, based on a comparison of the level of fluorescence in the tubes with the level of fluorescence in the background tubes of the corresponding set of reagents. The analysis results are displayed on the computer screen in the form of a table. A significant increase in the level of fluorescence was revealed, i.e., the presence of specific PCR products in a test tube containing a set of primers and fluorescently labeled probes complementary to the 35S promoter nucleotide sequence and the analyzed DNA.

Therefore, as a result of the analysis of beans of GM soybean line 40-3-2 detected transgenic DNA.

The results of the invention

The main advantage of the invention is the absence of the need to transfer the amplification after PCR to the cells of biochips. In the present invention, PCR amplification is carried out using a new set of oligonucleotides (table 1), including specific primers and fluorescently labeled probes, and PCR results are detected in closed amplification tubes using a fluorescent PCR detector, which completely eliminates the possibility of contamination of reagents and equipment with PCR products and prevents the receipt of false analysis results.

In addition, the proposed invention is high-tech, does not require significant labor costs. Pre-prepared tubes with a paraffin-sealed mixture for amplification are stored for a long time (more than six months) at room temperature, which allows them to be used as part of ready-made diagnostic kits. If necessary, it is possible to use additional sets of primers and fluorescently labeled probes to study a wider range of transgenic DNA sequences used to create transgenic plants.

The proposed invention does not pose a risk to the health of personnel and the environment, as it is not associated with the use of toxic or radioactive compounds.

Claims (1)

A method for identifying transgenic DNA sequences in plant material and products based on it, including DNA analysis using polymerase chain reaction (PCR) using a set of oligonucleotides - specific primers and fluorescently labeled probes complementary to these sequences and detection of PCR results, characterized in that PCR amplification is carried out using the set of oligonucleotides shown in Table 1, with the detection of the results of amplification in closed amplification tubes.