阅读说明：本技术 基于重组酶的环介导扩增方法 (Recombinase-based loop-mediated amplification method ) 是由 唐卓 陈刚毅 董娟 于 2019-11-18 设计创作，主要内容包括：本发明属于分子生物学技术领域,提供一种基于重组酶和单对引物的环介导扩增哑铃状结构形成方法及由此形成的新的核酸环介导扩增的方法(RALA)。本发明仅依赖重组酶和单对引物即可实现环介导扩增哑铃状结构的形成,具体为利用重组酶和一对单链引物形成的复合物打开双链DNA后使引物与模板链互补配对,通过聚合酶的延伸及单链置换作用获得哑铃状结构,依赖形成的哑铃状结构便可引发下游的环介导扩增。该方法引物设计简单,可有效控制体系复杂度和非特异扩增。(The invention belongs to the technical field of molecular biology, and provides a method for forming a ring-mediated amplification dumbbell-shaped structure based on a recombinase and a single pair of primers and a novel nucleic acid ring-mediated amplification method (RALA) formed by the method. The invention can realize the formation of the ring-mediated amplification dumbbell structure only by a recombinase and a single pair of primers, and particularly, the dumbbell structure is obtained by opening double-stranded DNA (deoxyribonucleic acid) by a complex formed by the recombinase and a pair of single-stranded primers, extending polymerase and performing single-stranded displacement, and downstream ring-mediated amplification can be initiated by depending on the formed dumbbell structure. The method has simple primer design and can effectively control the system complexity and non-specific amplification.)

1. A method for forming a ring-mediated amplification dumbbell-shaped structure based on a recombinase and a single pair of primers is characterized by comprising the following steps: opening double-stranded DNA by using a complex formed by a recombinase and a pair of single-stranded primers, complementarily pairing the primers and a template strand, and obtaining a dumbbell-shaped structure through extension of polymerase and single-stranded displacement; the 3 'end segment of the single-stranded primer is complementary to the sequence of the template, and the 5' end segment is complementary to the sequence of the downstream segment after the primer is extended.

2. The method for forming the ring-mediated amplification dumbbell structure based on the recombinase and the single pair of primers according to claim 1, which is characterized in that: the length of the segment of the single-stranded primer which is matched with the template at the 3' end is not less than 16 nucleotides.

3. The method for forming the ring-mediated amplification dumbbell structure based on the recombinase and the single pair of primers according to claim 1, which is characterized in that: the working principle is that a pair of primers (forward primer and reverse primer) is designed aiming at a target sequence to be amplified in the double-stranded DNA, the primers are composed of two parts, a 3 'end segment is complementarily paired with a template, a 5' end segment is complementary with a downstream segment after extension, in the presence of recombinase, a forward single-stranded primer (reverse primer) is combined with the recombinase to form a complex, the complex scans the double-stranded DNA, when a region homologous with the 3 'end segment of the primer is encountered, the double-stranded DNA is untied, the 3' end of the primer is paired with a complementary strand thereof, under the action of polymerase with strand displacement activity, one strand of the template is displaced while the 3 'end of the primer is extended, the double-stranded DNA formed by primer extension can be recognized by a reverse primer (forward primer) -recombinase complex, and the 3' end of the reverse primer (forward primer) is invaded into the double-stranded DNA template to be extended in the same way to form new short double-stranded DNA (forward primer, the reverse primer amplified fragment comprises a forward primer 5 ' end fragment and a reverse primer 5 ' end fragment), newly formed short double-stranded DNA is identified by a reverse primer (forward primer) -recombinase complex, a single-stranded DNA is displaced after the 3 ' end of the reverse primer (forward primer) is invaded and extended, the 5 ' end fragment (the sequence of which is the same as that of the reverse primer 5 ' end fragment) of the single-stranded DNA is complementary with a downstream sequence to form a stem-loop structure, and meanwhile, the 3 ' end fragment (the sequence of which is complementary with that of the forward primer 5 ' end fragment) is complementary with an upstream sequence to form a stem-loop structure, so that a dumbbell-shaped structure with stem-loops at two ends can be formed.

4. A recombinase and single primer-based loop-mediated amplification method (RALA), wherein downstream loop-mediated amplification is initiated by the dumbbell structure formed by the recombinase and single primer-based loop-mediated amplification dumbbell structure formation method of claim 1, thereby amplifying and detecting target sequence information.

5. The recombinase and single-pair primer-based loop-mediated amplification method (RALA) according to claim 4, wherein the operation principle is that the 3 ' end of the dumbbell-shaped structure is extended by itself as a template to complement the stem-loop structure at the 5 ' end into a double-stranded structure to form a long double-stranded structure with a single-stranded loop, at this time, the primer can be extended by pairing with the single-stranded loop sequence to displace the self-extended product and form a new stem-loop structure at the 3 ' end, the new stem-loop structure is extended by itself as a template to form a longer double-stranded DNA with a single-stranded loop, and the displaced single-stranded DNA forms a new dumbbell-shaped structure complementary to the previous dumbbell-shaped structure, and the two dumbbell-shaped structures are alternately generated and simultaneously form a large number of double-stranded DNAs with single-stranded loops which are repeated in series; additional loop primers can also be designed to accelerate the entire amplification process for a large number of single-stranded loop sequences generated during the amplification process, thereby achieving efficient amplification of the target sequence.

6. The method for forming a dumbbell structure according to claim 1 or the method for loop-mediated amplification (RALA) according to claim 4, which comprises: the recombinase used is an enzyme that can form a complex with a single-stranded DNA, recognize and unbind a double-stranded DNA homologous to the single-stranded DNA in the complex, and complementarily pair the single-stranded DNA with one of the template strands.

7. The method for forming a dumbbell structure according to claim 1 or the method for loop-mediated amplification (RALA) according to claim 4, which comprises: the polymerase used is a polymerase having neither 5 '-3' exonuclease activity nor 3 '-5' exonuclease activity, but strand displacement activity.

8. The method for forming a dumbbell structure according to claim 1 or the method for loop-mediated amplification (RALA) according to claim 4, which comprises: the reaction temperature is isothermal, and can be any constant temperature in the range of 37-70 ℃.

Technical Field

The invention belongs to the technical field of molecular biology, relates to a nucleic acid amplification method, and particularly relates to a method for generating a dumbbell-shaped structure intermediate by opening double-stranded DNA (deoxyribonucleic acid) for extension by using recombinase and a single pair of primers and a method for realizing nucleic acid loop-mediated amplification.

Background

In the field of nucleic acid detection, it is generally necessary to determine whether a particular gene sequence or a portion of a gene fragment is present in a sample to be detected. Since nucleic acid sequences in a sample are usually present in minute amounts, it is often necessary to amplify the target sequence in order to achieve detection. Currently, the most widely used method for nucleic acid amplification is the Polymerase Chain Reaction (PCR), and the exponential amplification method has the advantage of high sensitivity. However, the PCR method also has the following significant problems: depending on large-scale temperature control instruments, the method needs to be carried out in a professional laboratory.

In response to the above-mentioned disadvantages of the PCR method, many isothermal amplification methods of nucleic acids such as LAMP (Loop-mediated isothermal amplification), RPA (recombinase polymerase amplification), RCA (Rolling circle amplification), NASBA (nucleic acid dependent amplification detection technology), EXPAR (isothermal index amplification), HDA (isothermal index amplification), SDA (Strand Displacement amplification), CPA (Cross-primer isothermal amplification), MDA (multiple Displacement amplification) have now been developed. The isothermal amplification method has small dependence on precise temperature-changing equipment, does not need a fixed reaction site, and has wide application prospect in the field of POCT. For these known isothermal amplification methods, some methods require high-temperature pre-denaturation before the isothermal step, and others have problems in that primer design or reaction components are complicated. For example, in SDA (strand displacement amplification) methods, the system other than the polymerase is first denatured at 90 degrees or more and then the polymerase is added for amplification. In the RPA (recombinase polymerase amplification) method, the operation of the whole system requires the close coordination of 5 enzymes (UvsX, UvsY, gp32, Bsu polymerase, creatine kinase), and in addition, the addition of ATP and creatine phosphate is required to continuously supply energy to the system. In the LAMP (loop-mediated isothermal amplification) method, in order to form a dumbbell-shaped structure, two pairs of primers are required to be designed aiming at 6 regions of a template to realize amplification, the number of primers is large, the design difficulty and the system complexity are increased, and non-specific amplification is easy to occur.

Therefore, the development of an isothermal amplification technology with simple operation, simple system and high amplification efficiency is of great significance to the popularization of gene detection.

Disclosure of Invention

The invention combines recombinase and loop-mediated amplification technology, and provides an isothermal amplification method which has simple design, simple and convenient operation, high amplification efficiency and low instrument dependence.

The technical scheme for realizing the purposes is as follows:

a method for forming a ring-mediated amplification dumbbell-shaped structure based on a recombinase and a single pair of primers has the technical scheme that: opening double-stranded DNA by using a complex formed by a recombinase and a pair of single-stranded primers, complementarily pairing the primers and a template strand, and obtaining a dumbbell-shaped structure through extension of polymerase and single-stranded displacement; the 3 'end segment of the single-stranded primer is complementary to the sequence of the template, and the 5' end segment is complementary to the sequence of the downstream segment after the primer is extended. The specific working principle is as follows: a pair of primers (a forward primer and a reverse primer) is designed aiming at a target sequence to be amplified in DNA, the primers are composed of two parts, a 3 'end segment is complementarily paired with a template, and a 5' end segment is complementary with a downstream segment of the extended sequence. In the presence of recombinase, the forward primer (reverse primer) binds to the recombinase to form a complex, the complex scans the double-stranded DNA, when a region homologous to the 3 ' end fragment of the primer is encountered, the double-stranded DNA is cleaved, the 3 ' end of the primer is paired with its complementary strand, one strand of the template is displaced while the 3 ' end of the primer is extended by polymerase having strand displacement activity, the double-stranded DNA formed by primer extension can be recognized by the reverse primer (forward primer) -recombinase complex, and the 3 ' end of the reverse primer (forward primer) is invaded into the double-stranded DNA template in the same manner to be extended to form new short double-stranded DNA (forward and reverse primer-amplified fragments including the 5 ' end fragments of the forward and reverse primers), and the newly formed short double-stranded DNA is recognized by the reverse primer (forward primer) -recombinase complex, after the 3 ' end of the reverse primer (forward primer) is invaded and extended, a single-stranded DNA is displaced, the 5 ' end segment (the sequence is the same as that of the 5 ' end segment of the reverse primer) of the single-stranded DNA is complementary with a downstream segment of sequence to form a stem-loop structure, and meanwhile, the 3 ' end segment (the sequence is complementary with that of the 5 ' end segment of the forward primer) is complementary with an upstream segment of sequence to form a stem-loop structure, so that a dumbbell-shaped structure with stem loops at two ends can be formed.

The method for forming the ring-mediated amplification dumbbell structure based on the recombinase and the single pair of primers can be applied to all amplification methods which depend on the dumbbell structure to generate the ring-mediated amplification, thereby forming a novel ring-mediated amplification method (RALA).

The invention relates to a recombinase and single-pair primer-based loop-mediated amplification method (RALA), which is used for initiating downstream loop-mediated amplification by utilizing a loop-mediated amplification dumbbell-shaped structure formed by the recombinase and the single-pair primer to amplify and detect target sequence information. Firstly, extending the 3 ' end of the dumbbell-shaped structure by taking the dumbbell-shaped structure as a template, filling the stem-loop structure at the 5 ' end into double chains, and forming a long double-chain structure with a single-chain loop, wherein the primer can be matched with the single-chain loop sequence to extend and replace a self-extending product and form a new stem-loop structure at the 3 ' end, the new stem-loop structure is extended by taking the primer as a template to form longer double-chain DNA with the single-chain loop, and the replaced single-chain DNA forms a new dumbbell-shaped structure with a sequence complementary with the previous dumbbell-shaped structure, and a large amount of double-chain DNA with the single-chain loop which is repeated in series is formed when the two dumbbell-shaped structures are alternately generated. Additional loop primers can also be designed to accelerate the entire amplification process for a large number of single-stranded loop sequences generated during the amplification process, thereby achieving efficient amplification of the target sequence.

The recombinase of the invention is an enzyme which can form a complex with a single-stranded DNA, recognize and unwind a double-stranded DNA homologous to the single-stranded DNA in the complex, and complementarily pair the single-stranded DNA with one of the template strands.

The polymerase of the present invention is a mesophilic polymerase having neither 5 '-3' exonuclease activity nor 3 '-5' exonuclease activity, but strand displacement activity.

The method for forming the ring-mediated amplification dumbbell structure based on the recombinase and the single pair of primers and the corresponding ring-mediated amplification method (RALA) can form the dumbbell structure only by one pair of primers, the primers are simple in design, and the system complexity and the risk of non-specific amplification can be effectively reduced.

The method of the invention can detect double-stranded DNA and single-stranded DNA.

The length of the fragment at the 3' end of the primer of the invention can not be less than 16 nucleotides.

The reaction temperature of the method is isothermal, and can be any constant temperature within the range of 37-70 ℃.

As used herein, the following words/terms have the following meanings, unless otherwise specified.

"DNA": deoxyribonucleic acid. Is a biological macromolecule with genetic information, is formed by connecting 4 main deoxyribonucleotides through 3 ', 5' -phosphodiester bonds, and is a carrier of the genetic information.

"PCR": polymerase chain reaction. The method is a method for synthesizing specific DNA fragments in vitro by enzyme, and is carried out in a cycle consisting of a plurality of steps of reaction such as high-temperature denaturation, low-temperature annealing, suitable temperature extension and the like, so that the target DNA can be rapidly amplified, and the method has the characteristics of strong specificity, high sensitivity, simple and convenient operation, time saving and the like.

"target sequence": the analytes to be detected include DNA and RNA sequences.

The "complex": the recombinase forms a complex with the single-stranded DNA in a form similar to that of the double-stranded DNA, and winds around the single-stranded DNA to extend spirally.

"mesophilic" or "mesophilic polymerase": relative to a thermophilic enzyme such as Taq DNA polymerase. Here, mesophilic enzyme refers to an enzyme that does not tolerate high temperatures in the operating temperature range of 15-70 deg.C, such as Bsm DNA polymerase (Thermo Fisher)^TM30-63 ℃), Bst DNA polymerase (NEB,<70 ℃ C.), T4 DNA ligase (NEB, recommended reaction temperature 16 ℃ C., 20-25 ℃ C.), T4 polynucleotide kinase (NEB, recommended optimum reaction temperature 37 ℃ C.), etc.

"isothermal" or "isothermal conditions": the working temperature of the mesophilic enzyme used may refer to a constant temperature condition in the working temperature range of the mesophilic enzyme, or may refer to a temperature condition that dynamically changes in the working temperature range.

The key point of the method disclosed by the invention is that a recombinase and a single pair of primers are utilized to recognize and unwind double-stranded DNA and extend the double-stranded DNA to obtain a dumbbell-shaped structure so as to initiate the downstream loop-mediated amplification reaction. Both single-stranded and double-stranded DNA can be used as templates for this amplification method. The method has the advantages of simple primer design and high amplification reaction efficiency. The invention has obvious advantages over the prior art, and the main advantages thereof comprise:

1. is novel. The existing LAMP technology is complex in primer design, and many primers are easy to cause non-specific amplification. The invention combines the capacity of recombinase for homologous recognition of double-stranded DNA, avoids the dependence of loop-mediated amplification on external primers, can complete the formation of a dumbbell structure and the downstream loop-mediated amplification by only one pair of primers, has simple primer design, and can effectively reduce the system complexity and the risk of non-specific amplification.

2. And (4) universality. The RALA amplification method provided by the invention can realize amplification by only reasonably designing primers aiming at different templates, and is a universal method.

3. And (5) practicability. All reaction components are simple and easy to obtain, the reaction is carried out under the isothermal condition, no complex temperature control equipment is relied on, the reaction is rapid and efficient, and the method is suitable for amplification analysis of almost all nucleic acid templates and has great practical value particularly in the field of POCT.

4. And (4) economy. The reagents, proteins and enzymes involved in the invention are widely available and readily available.

Drawings

FIG. 1 is a schematic diagram of a method for forming a dumbbell structure based on a recombinase and a single pair of primers and a corresponding loop-mediated amplification method (RALA) according to the present invention.

FIG. 2 is a graph showing the results of example 1.

FIG. 3 is a graph showing the results of example 2.

FIG. 4 is a graph showing the results of example 3.

Detailed Description

The invention will be further illustrated by way of example with reference to the accompanying drawings. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.