RU2375458C2 - Method for transgene copy count in dna sample - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention concerns molecular genetics. There is disclosed method for transgene copies count in DNA sample. The method implies performing competitive two-array PCR including control sample and culture of copy number marker in one test tube. The amplification products are separated by electrophoresis in agarose gel. The electrophoregram is coloured with ethidium bromide in UV-light. Optical density of amplicon strips is measured with plotting a calibration curve whereon transgene copies in the sample is counted. The method is characterised with high sensitivity and accuracy.

EFFECT: invention can be used for manipulation with transgene organisms in laboratory experiments and biotechnological developments.

1 dwg, 1 tbl, 1 ex

Description

The invention relates to molecular genetics, in particular to methods for determining the copy number of transgenes, and can be used to analyze transgenic organisms in laboratory experiments and in biotechnological work.

A known method for determining the number of copies of a transgene, consisting in the fact that a DNA sample isolated from the tissue of a transgenic organism is subjected to Southern-blot hybridization with a radiolabeled probe. In parallel, hybridization is carried out with samples of a similar gene in which the copy number of the transgene is known. With hybridization tests, a radio autograph is performed. The number of copies of a transgene is judged by the intensity of the exposed spots on the radio autogram. (Nucleic Acids Res. 2006; 34 (8): 2154-2165. The human vitamin D-binding protein gene contains locus control determinants sufficient for autonomous activation in hepatic chromatin. Tomoko Hiroki, Young-Han Song, Stephen A. Liebhaber, and Nancy E. Cooke)

The disadvantages of this known method include low sensitivity and accuracy, difficulties in interpreting the results, as well as the need to use radioactive materials. This method is chosen by us as a prototype.

The objective of the invention is to increase the sensitivity and accuracy of determining the number of copies of a transgene.

The problem is solved due to the fact that the following differences are provided in the method for determining the number of copies of a transgene, including comparing a DNA sample isolated from a transgenic organism with DNA samples of known copy number:

primers for the experimental transgene are selected;

the copy number marker (MK) is synthesized — a DNA fragment having an insert in the amplified DNA fragment and the number of copies in vitro is determined;

carry out a series of competitive PCR with selected primers and two matrices - a prototype and various dilutions of the copy marker in one tube;

amplification products are separated by agarose gel electrophoresis and stained with ethidium bromide;

photograph the stained electrophoregram and measure the optical density of the amplicon bands;

build a calibration curve, which calculate the number of copies of the transgene.

The essence of the proposed method is as follows. Primers are selected for the transgene fragment. Then create a gene construct, in the composition of which there is an amplifiable fragment of the transgene, increased by insertion by 10-15%. Transgene primers should be suitable for the copy number marker. In this case, amplification products with selected primers when used as a matrix of transgene and copy marker will differ in size, which is easy to identify on the electrophoregram. Limitations on the size of the insert are due to the fact that an insert of insufficient size can make it difficult to distinguish amplification products on the electrophoregram, and an excessively large insert can lead to changes in the amplification conditions.

The amplification product of the gene fragment with an insert was used as a copy marker.

Having isolated and cleaned the copy marker and knowing its size and molecular weight, it is easy to calculate the concentration and number of copies of the copy marker in a unit volume by measuring the optical density with a spectrophotometer at a wavelength of 260 nm. To determine the number of transgene copies in the analyzed DNA sample of the transgenic organism, competitive PCR was performed in samples containing both the analyzed DNA sample (in the same concentrations) and the copy number marker in successive dilutions as matrices that competed for the common primers.

At the end of the PCR, the amplification products were separated by agarose gel electrophoresis with ethidium bromide and a digital image of the stained electrophoregram was obtained, which was processed with PhotoM 1.31 (http://t-lambda.chat.ru/download.html). With its help, the optical density of the amplicon bands was measured. We plotted where along the X axis are the lg values of the number of copies of the copy marker in samples, and on the Y axis, the ratio of the optical density of the amplicon band of the copy marker to the sum of the optical densities of both amplicons (copy marker and transgene). The number of copies of the transgene in the analyzed sample will be equal to the number of copies of the copy marker at Y = 0.5. This value is easy to find on the chart.

The use of PCR in the claimed method of reaction allows to significantly increase the sensitivity of the determination compared to the prototype due to the multiple doubling of the initial amplified fragments of the transgene. The high sensitivity of the method for determining the number of copies of a transgene is important in cases where it becomes necessary to analyze the microquantities of the starting material, for example, in the study of embryos and microorganisms.

Digital methods of processing electrophoregram images increase the accuracy of determination, make it possible to quantitatively measure the optical density of the colored bands of amplification products. The prototype involves procedures such as exposure of labeled samples with x-ray plates, followed by their manifestation and fixation. Each of these steps can also introduce distortions into the final result. Unlike the prototype, the method does not involve the use of radioactive materials, and therefore is safer and more convenient. The invention improves the sensitivity and accuracy of determining the number of copies of a transgene. The possibility of carrying out the claimed invention is confirmed by the following example.

Example

There was a plasmid vector containing the LacZ reporter gene. Mouse zygotes were transformed with this vector and transplanted into the uterus of the surrogate mother. At the stage of 10.5 days of development, the embryos were removed. Chromosomal DNA was isolated from amniotic membranes and analyzed for the presence of a transgene. The analysis was carried out by PCR using primers selected for the LacZ gene: direct 5'-acaggaaggccagacgcgaa-3 'and reverse 5'-ggcaacatggaaatcgctga-3'. The amplified fragment of the LacZ gene is 283 base pairs and has the AatII restriction endonuclease recognition site at the 234th base position.

The analysis revealed a transgenic embryo.

To determine the number of copies of a transgene in a transgenic DNA sample, an auxiliary genetic construct, a copy number marker, was created. For this, an insert of a double-stranded oligonucleotide of the following composition was ligated into the LacZ gene of the plasmid vector at the AatII site:

Due to the insertion during PCR with the same primers using the copy marker as a matrix (compared with the used transgene), the amplified fragment increases by 30 base pairs and amounts to 312 base pairs.

Marker copy (MK) was selected, cleaned. Its concentration (6 μg / ml) was determined on a spectrophotometer.

The total length of the amplicon with the insert is 312 bp

The molecular weight of the amplicon is 202488

The calculation of the number of copies of MK was carried out using the online service

http://molbiol.ru/scripts/01 07.html:

- 1 mol, 6 × 10 ²³ copies (Avagadro number) - weigh 202488 g

- or 6 × 10 ²³ copies weigh 202488 × 10 ⁶ mcg

- 1 ml MK contains 6 mcg

- Therefore, 1 ml of MK contains 17844153727630 copies of molecules.

Put competitive PCR with selected primers. Each sample contained two matrices: a DNA sample of a transgenic embryo and a copy number marker at various concentrations. The composition of the reaction sample before making the copy marker is shown in the table.

Table Reagent Final concentration Sterile deionized water up to a volume of 20 μl 10 × Taq Sintol Buffer 1 × 2 mM dNTP "Fermentas" 0.2 mm Direct primer 0.4 μM Reverse primer 0.4 μM Taq DNA polymerase 5 U / μl Termostar "Sintol" 2 E 25 mM MgCl ₂ 2.5 mm DNA matrix of embryo No. 18 45 ng

The copy marker was added to the samples in the amounts: 28, 56, 140, 279 and 560 copies per sample. Amplification mode: 95 ° - 5 min, (95 ° - 30 sec, 57 ° - 30 sec, 72 ° - 30 sec) - 32 cycles, 72 ° - 5 min.

Amplification of the samples was carried out on a Thermocycler "Tetsik". At the end of the reaction, 10 μl of the reaction mixture was added to the wells of a 2% ethidium bromide agarose gel and electrophoresis was performed. The image of the electrophoregram in UV light was obtained on the device "BIOSKAN" (figure 1). Using the program PhotoM 1.31 measured the optical density of the bands of amplicons in the inverted image of the electrophoregram, and then built a calibration graph (figure 2).

Equal number of copies of MK and transgene in the sample occurs when the ordinate value of the graph is 0.5. According to the graph at Y = 0.5, the value of X (lg of the number of copies) is 2.02, therefore, the number of copies of the transgene in each of the samples is 105.

Brief Description of the Drawings

Figure 1. Inverted image of an electrophoregram in UV light of competitive PCR amplification products.

2 - leading amplicon, from the matrix of the LacZ gene of the transgenic embryo;

1 - MK is the matrix for the lagging amplicon.

Figure 2. Calibration chart for calculating the number of copies of a transgene in a DNA sample.

The ordinate axis is the ratio of the optical density of the transgene amplicon to the total optical density of MK amplicons and the transgene on the electrophoregram.

The ordinate is the decimal logarithm of the number of copies of MK in the sample.

Claims (1)

A method for determining the number of copies of a transgene in a DNA sample, comprising comparing a DNA sample isolated from a transgenic organism with known copies of DNA, characterized in that primers for the experimental transgene are selected, a copy marker is synthesized — a DNA fragment having an insert in an amplified DNA fragment, and determine its copy number, conduct competitive PCR with selected primers and two matrices - a prototype and various dilutions of the copy marker, in one test tube, amplification products are separated by ektroforezom agarose gel and stained with ethidium bromide, and photographed colored electrophoretogram measured absorbance bands amplicons construct a calibration graph that was calculated the number of copies of the transgene in the sample.

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