RU2410439C1 - Method for ablation of target dna from surface of dna biochips - Google Patents

Abstract

FIELD: chemistry; biochemistry.

SUBSTANCE: invention pertains to biotechnology, laser technology and medicine. disclosed is a method for ablation of target DNA from the surface of DNA biochips, involving immobilisation of a DNA sample on a substrate, hybridisation of the immobilised DNA sample with the target DNA, ablation of the target DNA from the hybridised biochip through exposure to laser radiation and then determining the structure of the ablation products via polymerase chain reaction (PCR). Ablation is carried out with terahertz laser radiation on free electrons with wavelength 120-135 mcm, intensity 20-40 W/cm², pulse repetition frequency 5.6 MHz, pulse duration of 50 ns with minimal width ratio of exposure line of 3×10^-3. The substrate used is in form of pre-polished and activated plates made from high-resistivity silicon or porous aluminium oxide silufol.

EFFECT: method provides non-destructive DNA soft ablation and enables analysis of target DNA from the specific primary structure and functional biological activity.

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Description

The invention relates to biotechnology and medicine and can be used to increase the efficiency (effectiveness, accuracy, quality) of analytical and diagnostic operations using solid substrates (chips) with deoxyribonucleic acids (DNA) deposited on them (DNA biochips).

In recent years, the number of studies using biochips (gene arrays), which are small-sized glass, polymer, ceramic plates or nylon membranes carrying biopolymers on their surface, such as proteins, DNA, DNA fragments, has grown avalanche-like.

A significant advantage of biochips in comparison with traditional methods of biological material analysis is the possibility of simultaneous analysis of a huge (up to 100 thousand) number of samples. The appearance of DNA biochips made it possible to use them for the comprehensive diagnosis of human genetic, infectious and oncogenic diseases in medical institutions. The ability to determine the expression level of 42 thousand human genes and proteins allows biotechnological companies to use biochips to develop and test new drugs for the rapid detection of pathogenic bacteria and viruses in the human body.

In recent years, the amount of data on the use of biochips in scientific and clinical research has increased exponentially due to the high efficiency of their use in real practice. Accordingly, the number of manufacturers of biochips and methods for their production has grown significantly. The need to compare the results of analysis in different experiments between different laboratories and databases has become very urgent. In this regard, to date, many researchers are actively interested in comparing different platforms and analyzing the results of using biochips of different manufacturers on identical nucleic acid mixtures (Harbig J, Sprinkle R, Enkemann SA. A sequence-based identification of the genes detected by probesets on the Affymetrix U133 plus 2.0 array. Nucleic Acids Res 2005; 33 (3): e31). By 2003, it became clear that the results of the identification of DNA sequences on biochips of various manufacturers had a total convergence of only about 4% when analyzing identical DNA mixtures (E. Marshall SCIENCE 2004, V.306, 22, OCTOBER, pp. 630-631). Researchers and manufacturers of biochips around the world have made tremendous efforts to solve this problem, and by 2007 the convergence of results had significantly increased to 23%. One aspect of the problem is that different biochip manufacturers use samples with different nucleotide sequences to identify the expression of the same gene. Using for comparison only that part of the samples that has a similar sequence, increases the convergence of the results, but significantly limits the information content of the analysis. Only constant monitoring of expression and subsequent application of the methods of statistical processing of gene set enrichment analysis (GSEA) and parametric analysis of gene enrichment (PAGE) allowed us to obtain acceptable repeatability of the results. Thus, the question of what kind of sequence actually hybridizes on the biochip is still relevant.

Today, in Russia, the market for biochips of domestic production is being formed. For the first time in the world in Russia biochips have been developed for the rapid identification of drug-resistant forms of tuberculosis. Work is underway to create biochips for typing the influenza virus, testing helminthiases, identifying and identifying individuals, and to study the specificity of DNA-protein interactions.

However, there is still no opportunity to directly compare the results of hybridization of target DNA in microcells of biochips. This leaves open the problem of identification of target DNA hybridized into microcells of biochips. Therefore, the task of diagnosing the identity of DNA biochips of different production, created using similar technologies, is relevant.

Currently, for the ablation (removal) of biopolymers from the surface of chips, in particular biochips, various chemical, physical and physicochemical methods are used. In patent application JP 2004212215 A, op. 07/29/2004 describes a method of ablation from the surface of biochips, such biopolymers as nucleic acids, peptides, polysaccharides, by means of focused laser pulsed radiation followed by analysis of biopolymers on a quadrupole mass spectrometer. A similar approach is used in patent application US 20030469160 A1, op. 02/21/2002 for the ablation of RNA, DNA, peptides and sugars by ultrashort pulses of laser radiation for ionization and transfer of biopolymers to the atomic state with subsequent mass spectrometry. In the patent application US 2005185260 A1, op 24.02.2005, describes the use of an optical fiber device for irradiating biochips with picosecond and nanosecond pulsed terrahertz radiation and ablation of biopolymers. In patent application US 2003180720 A1, op. 09/25/2003, for the ablation of biomolecules, in particular proteins and nucleic acids, from the surface of biochips, Raman spectroscopy is used. In patent application US 2003162216 Al, op. 08/28/2003 for the analysis of the studied proteins (cells, viruses, bacteria) conjugated from the biochip surface, as well as DNA, hormones, sugars, cofactors, metabolic products, drugs and toxins, reagents specific to each object are used. In patent application US 2006443639 A1, op. 05/30/2006 a method for binding biopolymers to the surface of biochips using terrahertz and gigahertz radiation with a wavelength of 3 mm to 3 μm and a pulse repetition rate in the range from 0.1 to 100 THz is described. The binding of the analyzed biomolecules to the biochip, on the surface of which a specific binding component is immobilized, is detected by the ratio of the intensity levels of the vibrational spectra of gigahertz and terahertz radiation before and after the formation of biomolecular complexes. The method allows with high accuracy to fix the specific binding of the analyzed biomolecules.

All the described methods of ablation of biological macromolecules (biopolymers) from the surface of solid substrates (biochips), including laser ones, do not allow detection of the studied objects by their specific primary structure, and functional biological activity (enzymatic, regulatory, receptor, etc.) biopolymers are completely lost due to the “hard” effect on functional macromolecules, leading to a change in the conformation of macromolecules, active centers, secondary and tertiary structures.

Closest to the claimed method, the prototype is a method of ablation of biopolymers from a solid substrate using an excimer laser (ProteinChip Reader Model PBS II) with subsequent determination of the structure and specific activity of ablation products (He Z, Zhong H, Hu Y, Xiao S, Xu J Analysis of differential protein expression in Acidithiobacillus ferrooxidans grown under different energy resources respectively using SELDI-ProteinChip technologies, Anal Biochem. 2005 May 15; 340 (2): 317-29). However, the known method does not allow non-destructive "soft" ablation of the biopolymer.

The disadvantage of the prototype, as well as all known laser methods of ablation of biopolymers, is that they do not allow to achieve “soft” ablation from the surface of the biochip of biopolymers while maintaining their structure and specific biological functional activity, which reduces the information content and quality of analysis of biological objects with specific activity (enzymatic, regulatory, receptor) is the main criterion for their biospecificity.

The modern scientific and technical literature does not describe methods for “soft” ablation of biopolymers, in particular DNA, from the surface of solid substrates, including biochips, which allow preserving specific types of their biological activity.

The technical task of the present invention is to expand the functionality of the method and increase its information content.

The stated technical problem is achieved by non-destructive ("soft") ablation of the target DNA from the surface of the DNA biochip using terahertz radiation of a free electron laser (FEL) with a wavelength of 120-135 μm.

The essence of the proposed method is as follows. A DNA sample is immobilized on a polished and activated plate of high-resistance crystalline silicon or aluminum oxide, the immobilized DNA sample is hybridized with the target DNA (sample), then the target DNA is softly ablated from the hybridized biochip with subsequent evaluation of the results of soft ablation using PCR. In this case, DNA is ablated by terahertz radiation of a free-electron laser with a wavelength of 120-135 μm, with an intensity of 20-40 W / cm ² , a pulse repetition rate of 5.6 MHz, a pulse duration of 50 ns with a minimum relative exposure line width of 3 × 10 ^-3 .

Ablation using terahertz radiation of FEL is the transfer of molecules into the aerosol phase from a solid or liquid phase without changing the molecular structure, while maintaining the complete identity of the nucleotide sequence of the original target DNA and the oligonucleotide ablated after hybridization.

To conduct “soft” laser ablation of biomacromolecules without their destruction, an analyzer was used to analyze DNA biochips.

Figure 1 presents a schematic diagram of the action of such an analyzer. Under the influence of radiation, hydrogen bonds stabilizing the DNA-DNA hybrid molecules are destroyed and the DNA probe ablates and enters a stream of nitrogen. A DNA probe trapped in a nitrogen stream is collected on a trap membrane and then analyzed by PCR.

To confirm the effectiveness of “soft” laser DNA ablation by electrophoretic methods, individual DNA fragments of lambda phage hydrolyzed by HindIII restriction enzyme were preparatively isolated and soft laser ablation of DNA fragments of 3000 nucleotide pairs (np), 9000 np and 23000 np was performed.

Figure 2 presents the measurement results of the diffusion size of aerosol particles obtained by soft ablation under the influence of THz radiation with a wavelength of 128 microns by the claimed method.

From figure 2 it is seen that the diffusion size of the particles obtained by exposure to THz radiation, quite well correspond to the linear dimensions of the polymer chains of DNA taken in the experiment. The data obtained indicate that the method of non-destructive soft ablation is applicable to the analysis of DNA hybrids.

Thus, the possibility of “soft” laser ablation of DNA molecules while maintaining their molecular structure has been shown for the first time.

The invention is illustrated by the following examples of specific performance.

Example 1

A) Activation of the silicon surface of the substrate (chip)

A round plate with a diameter of 20 mm and a thickness of 1 mm and a plate of high-resistance (6-8 kOhm / cm) silicon were used as a substrate for immobilizing DNA samples.

After manufacturing, the wafer was polished, since a polished silicon wafer does not give aerosol particles during ablation and has a high transmittance in the working region of the FEL emission spectrum, therefore, it heats up little during irradiation and does not lead to thermal degradation of the biopolymer.

To activate the silicon surface of the chip (creating an epoxy coating), the silicon wafer was soaked in a solution of 0.1% GPS (glycidoxypropyltrimethoxysilane) in toluene (Aldrich, 244511-1L) for 30 min at 40 ° C, washed three times in toluene and then dried at 110 ° C.

B) Immobilization of DNA samples on a substrate (chip)

To fix the DNA sample on the surface of the chip, an oligonucleotide of a given length with a covalently linked linker molecule to the 5'-end was used.

Sample Structure: Linker (L1) -5'-ATATCAGATCGCAGTGT-3 '

Figure 3 presents the chemical structure of the branched linker 1.

A solution of the DNA sample (2 nanomoles in 2 μl 3 × SSC) was applied to the activated surface of the chip, in the central part. After applying the sample to the epoxy surface, the following procedure was used to increase its immobilization efficiency: the chip in a Petri dish was left overnight in a desiccator with saturated NaCl solution (to ensure 50% humidity) at 42 ° C, then washed in 0.2% SDS 2 min on a shaker (Eppendorf) and then washed three times with bidistilled water, incubated in bidistilled water (50 ° C) for 20 minutes and dried.

C) Hybridization of the sample with a sample, washing and drying the chip

As a target DNA (sample 1), a DNA fragment of 50 nucleotides in length with a fluorescent label at the 5'-end was used. Fluorescein-6 (FAM6) was used as a fluorescent dye (label). The structure of the hybridizable sample 1:

5'-6FAM-TCCCTCCTGAGTTCCCCTGCACACACAACGGCACACTGCGATCTGATAT-3 '

The hybridization buffer contained a 3 × SSC solution with the addition of 0.5% SDS. 2 nanomoles of sample 1 were used for hybridization. Hybridization was performed overnight in a Corning hybridization chamber at 50 ° C with a gradual decrease in temperature to 37 ° C.

After hybridization, the chips were washed in an aqueous solution of 1 × SSC 2 times for 30 s at 37 ° C. The chips were dried at room temperature.

D) Conducting mild ablation of the target DNA of a hybridized biochip.

The terahertz radiation generated by FEL with the following parameters was applied to the surface of a hybridized biochip with a target DNA molecule:

Wavelength, microns 120 Pulse Duration, ns fifty Pulse repetition rate, MHz 5.6 Intensity, W / cm ² twenty Minimum relative line width 3 · 10-3

After “soft” ablation, the target DNA molecule transferred to the aerosol phase by a stream of nitrogen was collected on a nitrocellulose filter (Millipore, 0.22 μm). The target DNA particles collected on the filter were eluted with 200 μl of TE buffer (10 mM Tris-HCl (pH 8), 1 mM EDTA (pH 8)), 15 minutes at 65 ° C, and the sequence of the collected DNA was determined by PCR.

E) Evaluation of the results of mild ablation using PCR

The following primers were used to identify the structure and integrity of the target DNA molecule, washed off the filter after the soft ablation procedure:

For: 5'-TTCCCTCCTGAGTTCCCC-3 '

Rev: 5'-ATATCAGATCGCAGTGTGCC-3 '

The following PCR conditions were used:

32 cycles in the mode: denaturation 0.5 min at 95 ° C; annealing for 0.5 min at 53 ° C; synthesis 0.5 min at 7 ° C. The reaction mixture with a volume of 15 μl contained: 1 μl of sample 1 with a concentration of 5 × 10 ⁵ molecules / μl, 0.25 μM primers, 0.2 mm each of dNTP deoxynucleoside triphosphates, PCR buffer (75 mm Tris-HCl (pH 9) , 1.5 mm MgCl ₂ , 20 mm (NH ₄ ) ₂ SO ₄ , 0.01% Tween-20) and 0.4 units. Taq polymerase. It was found that the nucleotide sequences of the original target DNA and the oligonucleotide ablated after hybridization are identical.

Example 2

The experiment was carried out analogously to example 1, except for the following.

A round, polished and activated porous alumina silufol plate with a diameter of 20 mm and a thickness of 1 mm was used as a substrate for applying the studied biopolymer.

As the target DNA (sample 2), a DNA fragment of 90 nucleotides in length was used, without a fluorescent label at the 5'end. The structure of the hybridizable sample 2:

5'TTCCCTCCTGAGTTCCCCTACACACACAACCACACACAACCACACACAACCACACACACACACACACACAACGGCACACTGCGATCTGATAT-3 '

As a DNA probe, a complementary to sample 2 oligonucleotide 17 np in length was attached to the surface of the biochip.

Sample Structure: Linker (L2) -5'-ATATCAGATCGCAGTGT-3 '

Figure 4 presents the chemical structure of the linker 2 with a linear structure.

The ablation of the target DNA from the surface of the hybridized biochip was performed using terahertz radiation generated on FEL with the following parameters:

Wavelength, microns 135 Pulse Duration, ns fifty Pulse repetition rate, MHz 5.62 Intensity, W / cm ² 40 Minimum relative line width 3 · 10-3

After “soft” ablation, the target DNA molecule transferred to the aerosol phase by a stream of nitrogen was collected on a nitrocellulose filter (Millipore, 0.22 μm). The target DNA particles collected on the filter were eluted with 200 μl of TE buffer (10 mM Tris-HCl (pH 8), 1 mM EDTA (pH 8)), 15 minutes at 65 ° C.

DNA ablated from the surface of the biochip was amplified by PCR. Then its sequence was determined using Big Dye® technology (Applied Biosystems). The results are presented in figure 5. Mutually complementary sequences of the target DNA (sample 2) and DNA samples are marked with a brace.

Figure 5 shows that the nucleotide sequence of the original target DNA and the oligonucleotide ablated after hybridization are identical.

Thus, as a result of mild non-destructive ablation, it was possible to transfer to the aerosol phase, to collect and amplify the target DNA (samples 1 and 2) from the biochip surface under the influence of terahertz radiation of FEL and to establish the identity of the structure of the target DNA ablated after hybridization.

The use of “soft” ablation of target DNA from the surface of biochips when using terahertz radiation generated by FEL allows you to preserve the original structure of the DNA molecule and increase the information content and quality of analyzes using biochips.

Claims (3)

1. The method of ablation of target DNA from the surface of DNA biochips, including the immobilization of a test sample on a substrate, specific binding of an immobilized test sample to a target sample, ablation of a target sample from a biochip by laser irradiation, followed by determination of the structure of ablation products, characterized in that the exposure is carried out by terahertz radiation of a free electron laser with a wavelength of 120-135 μm, an intensity of 20-40 W / cm ² . 2. The method according to claim 1, characterized in that the pre-polished and activated wafers of high-resistance silicon or porous alumina silufol are used as a substrate. 3. The method according to claim 2, characterized in that for activation, the plates are soaked in a solution of 0.1% GPS in toluene for 30 min at 40 ° C, washed three times in toluene and dried at a temperature of 110 ° C.

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