RU2806069C2 - Concentration-based dna sequencing device - Google Patents

Abstract

FIELD: biotechnology; genetic engineering.

SUBSTANCE: device for DNA sequencing, containing: a reaction chamber configured to receive a nucleic acid sample, contains a solid substrate carrying fragments of genomic DNA to be amplified, and the reaction chamber is equipped with cooling and heating systems of any type to regulate the temperature of the reaction chamber, a liquid transfer system for the appropriate delivery of all components for the addition of one nucleotide at each step of the sequencing reaction, a sensor of any type that can be placed in any part inside or outside the reaction chamber, providing information about the concentration of nucleotides before and after addition of each nucleotide, and a control system connected to the input unit and the output unit for corresponding programming, configured to control parts of the device, regulate the temperature of the reaction chamber and collect data obtained from sensors in the reaction chamber.

EFFECT: performing DNA sequencing based on changes in the concentration of added nucleotides before or after the reaction.

8 cl, 1 dwg

Description

Field of technology

This application relates to DNA sequencing methods based on measuring changes in the concentration of added nucleotides before and after a reaction.

State of the art

Sequencing principles:

Two main DNA sequencing methods have been previously developed:

1 - Sequencing by synthesis:

- Sanger method/chain termination method (Life Technologies, Applied Biosystems)

- pyrosequencing (Roche/454)

- sequencing based on reversible terminators (Illumina)

- third generation sequencing using zero-mode waveguides (Pacific Biosciences)

2 - Sequencing by ligation and detection of oligonucleotides

- SOLiD (Applied Biosystems)

Below is a more detailed description of these methods.

1 - Chain termination sequencing (Sanger)

This method uses a modified DNA replication reaction in which the growing strands are terminated by dideoxynucleotides (ddNTPs). Dideoxynucleotides (ddNTPs) lack the 3'-OH group required to form a phosphodiester bond. When an enzyme (DNA polymerase) is added, the primer is extended until the ddNTP is detected. The chain will end by enabling ddNTP. With the correct dNTP:ddNTP ratio, the chain breaks along the entire length of the template. All dangling strands are terminated by ddNTP added to this reaction. The resulting broken chains are separated by electrophoresis. A different dye or "color" is used for each of the four ddNTPs. Since terminating nucleotides can be distinguished by color, all four reactions can be performed in the same tube.

2 - Pyrosequencing

Each nucleotide is added in turn in each cycle. Then only one of the four generates a light signal. To prepare for the next cycle, the remaining nucleotides are removed enzymatically. The light signal is recorded on a pyrogram.

Pyrosequencing is based on the generation of a light signal by the release of pyrophosphate (PPi) upon addition of nucleotides.

- DNA _n +dNTP → DNA _n+i +PP _I

- PP _I is used to generate adenosine triphosphate (ATP) from adenosine pyrophosphate (APS).

- APS+PP _I → ATP

ATP and luciferase generate light by converting luciferin to oxyluciferin.

3 - Waveguide with zero mode

This method is real-time sequencing of a single molecule, based on detecting the identity of each nucleotide immediately after its incorporation into the growing DNA strand.

4 - Sequencing by ligation

Instead of a polymerase, a ligase is used, which joins probe sequences (DNA fragments instead of oligonucleotides). Upon addition of the probe, a fluorescent signal is generated. Based on fluorescence, the identity of the nucleotide can be inferred. This sequencing method includes more than one primer, which differs from the previous one by only one base.

5 - Nanopore sequencing

A nanoscale device is used that moves polymer molecules in a sequential monomer pattern through a very small volume of space. The device includes a detector that directly converts the characteristics of the moving polymer into an electrical signal. Transduction and recognition occur in real time, molecule to molecule. The device can examine thousands of different molecules in a few minutes and can examine very long stretches of DNA.

6 - Sequencing by synthesis (SBS)

Sequencing by synthesis involves detecting the identity of each nucleotide immediately after it is incorporated into the growing DNA strand in a polymerase reaction. SBS includes fluorescent in situ sequencing (FISSEQ) and pyrosequencing techniques.

Using a photocleavable linker, a different fluorophore is attached to each of the four bases. The DNA polymer aza includes a complementary single nucleotide analogue. The unique fluorescence detected depends on the included nucleotide. The fluorophore is then photochemically removed, the 3-OH group is chemically regenerated, and the cycle continues.

New in this application:

Sequencing principles and sequencing devices involve very expensive instruments and instruments. In addition, it is not easy to purchase such equipment in our laboratories. Most of these methods are complex and require the use of special enzymes or special labels, such as fluorescent dyes, which increases the cost of the experiments. Therefore, it is necessary to develop and implement new methods to simplify the technology and reduce the associated costs.

Description of the invention

New principle:

The term "DNA sequencing" is commonly applied to several methods and technologies that are used to determine the order of the nucleotide bases: adenine, guanine, cytosine and thymine in a DNA molecule. DNA sequencing is widely used in many application areas such as diagnostics, biotechnology, forensic biology and biological systematics, in the sequencing of the human genome and the Human Genome Project. In the proposed sequencing device, DNA sample fragments are amplified using the conventional PCR method. Individual nucleotides are added to the synthesized DNA. If the nucleotide is complementary to the DNA fragment being studied, changes in the concentration of the added nucleotide can be monitored by any method.

Detailed description of the device

Genomic DNA fragments are fixed on a solid support. Each DNA fragment is amplified by PCR to produce a DNA cluster. In the PCR method, the temperature of the reaction mixture must vary during the PCR cycle from 95°C to 40-60°C and finally to 72°C for a certain number of cycles. Each cluster, originating from a single DNA fragment, acts as one sequencing reaction. In a sequencing reaction, a DNA cluster is tested by adding one of the nucleotides at a time. If the nucleotide is complementary, the change in concentration of the added dNTP can be monitored. This change can be tested directly in any part of the sequencing chamber.

Detailed description of the sequencing chamber

1. A solid support carrying fragments of genomic DNA that must be amplified and then tested for complementary nucleotides. This chamber is equipped with electronic heating and cooling systems.

2. Each DNA fragment is amplified by PCR to obtain a DNA cluster. In the PCR method, the temperature of the reaction mixture must vary during the PCR cycle from 95°C to 40-60°C and finally to 72°C for a certain number of cycles.

3. Four different solutions, each containing all the necessary components to add one of the four types of nucleotides to the amplified DNA cluster, are placed in four different containers.

4. In a sequencing reaction, a DNA cluster is tested by adding one of the nucleotides at a time. If the nucleotide is complementary, the change in concentration can be monitored. This change can be tested directly in any part of the sequencing chamber or outside of it.

Brief Description of FIG. 1

1 - Reaction chamber (1) containing a solid support carrying fragments of genomic DNA to be amplified by any method.

2 - System (2) of heating and cooling of any type to control the temperature of the reaction chamber.

3 - A control unit for controlling all processes carried out in the reaction chamber (4), connected to a programming input unit and sensors (5), (6) inside or outside the reaction chamber, which collect data and send them to the control unit.

4 - Liquid and water transfer system (3) carrying all the necessary solutions for testing the DNA cluster for complementary nucleotides by adding one of the nucleotides at a time and washing.

5 - Measuring unit connected to sensors (5) and (6), including all types of sensors needed to check whether the nucleotide is complementary or not, and to detect any change in concentration that can be monitored. This change can be tested inside or outside the sequencing chamber. The data is then collected and can be sent to the main control unit.

Claims (13)

1. A DNA sequencing device containing: a) a reaction chamber configured to receive a nucleic acid sample contains a solid support carrying genomic DNA fragments to be amplified, wherein the reaction chamber is equipped with cooling and heating systems of any type to regulate the temperature of the reaction chamber, b) a fluid transfer system for the appropriate delivery of all components for the addition of one nucleotide at each step of the sequencing reaction, c) a sensor of any type that can be placed at any part inside or outside the reaction chamber, providing information about the concentration of nucleotides before and after the addition of each nucleotide, and d) a control system connected to an input unit and an output unit for appropriate programming, configured to control parts of the device, regulate the temperature of the reaction chamber and collect data received from sensors in the reaction chamber. 2. The device according to claim 1, characterized in that the control system is connected to an input unit for software development, a liquid transfer system, a reaction chamber, temperature control sensors and sequencing process sensors, which differ depending on the method used to detect a change in concentration by any method known in the art, and may be located inside or outside the reaction chamber. 3. The device according to claim 1, characterized in that the liquid transfer system removes unreacted nucleotide after measuring the nucleotide concentration at each step of the sequencing reaction. 4. The device according to claim 1, characterized in that the input unit is included for software development and parameter selection. 5. The device according to claim 1, characterized in that the control system regulates the temperature necessary for amplification of the sample before starting the sequencing reaction, and maintains the temperature for the corresponding addition of one nucleotide at each stage of the reaction of the reaction chamber, with cooling and heating systems of any type and the sensors are connected to the reaction chamber and connected to the control unit. 6. The device according to claim 1, characterized in that many control samples can be placed inside the reaction chamber for greater accuracy and reproducibility of the results. 7. The device according to claim 1, characterized in that the detection of changes in the concentration of the added nucleotide is carried out by a sensor of any type, and the sensors of the detection system can be located inside or outside the reaction chamber. 8. The device according to claim 1, characterized in that it does not specify a specific number of samples.

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