CN102816756B - Isothermal nucleic acid amplification reaction reagent and isothermal nucleic acid amplification method - Google Patents

Abstract

The invention discloses an isothermal nucleic acid amplification reaction reagent which comprises Tris buffer solutions, potassium acetate, magnesium acetate, dithiothreitol, polyethylene glycol, adenosine triphosphate (ATP), deoxy-ribonucleoside triphosphate (dNTPs), phosphocreatine, creatine kinase, a primer group, single-strand binding (SSD) protein, colon bacillus helicase RecQ protein, UvsY protein and DNA polymerase. The invention further discloses an isothermal nucleic acid amplification method which includes steps of extracting DNA or performing inverse transcription after DNA extracting, and adding the isothermal nucleic acid amplification reaction reagent and reacting the mixture at a temperature in a range between 25 DEG C and 45 DEG C for 10 minutes to 60 minutes to complete the nucleic acid amplification. Compared with traditional polymerase chain reaction (PCR) technologies and according to the technology, nearly no professional instrument is needed, the operation is simple, technical requirements for operators are low, and the reaction time only needs about half an hour.

Description

Isothermal nucleic acid amplification reaction reagent and isothermal nucleic acid amplification method

technical field

The present invention relates to a technology for isothermal nucleic acid amplification, specifically a high-sensitivity nucleic acid amplification that utilizes Escherichia coli helicase RecQ protein, UvsY protein, UvsX protein and recombinant Klewnow exo-polymerase to realize non-PCR instrument dependence technology.

Background technique

The use of polymerase chain reaction (full English name: Polymerase Chain Reaction, referred to as PCR) for in vitro nucleic acid amplification is a revolutionary technology developed since 1983, and has been widely used in modern agriculture, medicine and food industry, etc. field. Through this technology, the efficient amplification of trace amounts of nucleic acid can be achieved, and a very small amount of specific nucleic acid sequence molecules can be amplified to a level that can be detected by the instrument. PCR consists of three basic reaction steps: denaturation-annealing (annealing)-extension: ① Denaturation of template DNA: After the template DNA is heated to 90-95°C for a certain period of time, the template DNA is double-stranded or double-stranded by PCR amplification. The strand DNA dissociates to make it a single strand, so that it can combine with the primer and prepare for the next round of reaction; ②Annealing (annealing) of the template DNA and the primer: After the template DNA is heated and denatured into a single strand, the temperature drops to 55 -60°C, the primers are paired with the complementary sequence of the template DNA single strand; ③Primer extension: DNA template-primer conjugate is under the action of DNA polymerase, at 70-75°C, using dNTP as the reaction raw material, the target sequence is The template, according to the principle of base pairing and semi-reserved replication, synthesizes a new semi-reserved replication strand complementary to the template DNA strand, and repeats the three processes of denaturation-annealing-extension to obtain more "semi-reserved replication strands" , and this new chain can become the template for the next cycle. It takes 2-4 minutes to complete a cycle, and the gene to be amplified can be amplified several million times in 2-3 hours. Using PCR technology, some transgenic plants can be positively screened: by amplifying the specific fragments of various inserted functional genes, it can be judged whether the plant is a positively transformed plant, and the information of whether it carries a functional gene can be obtained, so as to proceed to the next step— identification of its function.

Conventional nucleic acid in vitro amplification technology (PCR technology), due to the need to use expensive PCR equipment and consume a lot of power, its cost and application range are limited to a certain extent. With the rise of constant temperature in vitro nucleic acid amplification, the limitations of traditional amplification techniques have changed. In the past decade, some isothermal nucleic acid amplification techniques that make in vitro nucleic acid amplification easier and more convenient have been rapidly developed. Development, such as TMA technology (transcription-mediated nucleic acid amplification technology), SDA technology (strand displacement nucleic acid amplification technology), LAMP (loop-mediated nucleic acid amplification technology), HDA (helicase-dependent isothermal nucleic acid amplification technology) can amplify DNA under isothermal conditions. These technologies only need a temperature control device to maintain a reaction temperature, that is, 60-65°C, and can achieve efficient nucleic acid amplification, thereby getting rid of the dependence on PCR instruments that precisely control temperature changes. If nucleic acid amplification can be realized at lower temperature, or even at normal temperature, it will further simplify nucleic acid amplification technology and facilitate the wider application of such technology.

Contents of the invention

The purpose of the present invention is to provide a nucleic acid isothermal amplification technology capable of realizing nucleic acid amplification at a lower temperature, especially at room temperature, and with higher specificity and/or sensitivity.

The inventors of the present application have found through research that: RecQ protein, UvsY protein and UvsX protein can unwind the double strand of the template, stabilize the single strand and cause strand replacement between the primer and the template under isothermal conditions, thereby being able to replace the denaturation of traditional PCR (94 degrees) and annealing (50-60 degrees) steps, at the same time, nucleic acid is synthesized under the catalysis of DNA polymerase. In the presence of the single-strand binding protein SSB and the condensing agent polyethylene glycol, the chemical equilibrium in this reaction process tends to the synthesis of products, and this process can be repeated continuously to finally achieve efficient amplification of nucleic acids.

The invention provides a nucleic acid amplification reaction reagent, which comprises Escherichia coli helicase helicase RecQ protein, UvsY protein, UvsX protein and DNA polymerase.

Specifically, the components of the nucleic acid amplification reaction reagent of the present invention include: Tris (PH7.4) buffer, potassium acetate, magnesium acetate, dithiothreitol, polyethylene glycol (PEG), ATP, dNTPs, phosphoric acid Creatine, creatine kinase, primer set, SSB protein, E. coli helicase RecQ protein, UvsY protein, UvsX protein and DNA polymerase.

The proportioning of nucleic acid amplification reaction reagent of the present invention is preferably as follows:

Tris 25-75mM

Potassium acetate 25-75 mM

Magnesium acetate 5-15 mM

Dithiothreitol 2.5-10 mM

PEG 5-10% (w/v)

ATP 5-10 mM

dNTPs 1-2 mM

Creatine kinase 2.7-4.3μg/U

Phosphocreatine 25-50 mM

Primer set Each primer 10 μM

SSB protein 800 ng/μL

RecQ protein 10 ng/μL

UvsX protein 60 ng/μL

UvsY protein 50 ng/μL

DNA polymerase 50 ng/μL.

The pH value of the Tris solution is preferably 7.4, and the molecular weight of polyethylene glycol is preferably 15000-20000; the DNA polymerase is preferably a recombinant Klewnow exo-polymerase, and the recombinant Klewnow exo-polymerase is a recombinant Klewnow fragment that removes the 3' → 5' exocut Enzyme activity, through the mutation of individual bases, its 5' → 3' polymerase activity is enhanced.

Fluorescent dyes such as SYBR green Ⅰ, SYTO-13 or SYTO-82 can also be added to the reaction reagents so that the results can be visually observed with the naked eye after the amplification is completed.

The length of the primers in this reaction system needs to be about 30 nt to ensure the recognition and specificity during the strand replacement process, and the number of primers can be increased with the number of detection target genes. Tris in the reaction system is an efficient buffer for maintaining the pH of the reaction system. Appropriate concentrations of potassium acetate and magnesium acetate are necessary for the reaction to proceed. The RecQ protein is a helical protein from Escherichia coli, and the UvsX protein and UvsY protein are recombinant proteins from T4 and T6 phages. This replacement process requires ATP to provide energy. With the assistance of SSB protein, recombinant Klewnow exo-polymerase can achieve specific extension of DNA after strand replacement. Since ATP is regenerated under the catalysis of phosphocreatine, the chemical balance in this reaction process tends to the synthesis of products, and this process can be repeated continuously, so as to finally achieve efficient amplification of nucleic acids.

The present invention also provides a method for nucleic acid amplification in vitro, which includes the following steps: ① Extract DNA, or reverse transcribe after extracting RNA to obtain cDNA; ② Add the above-mentioned nucleic acid amplification reaction reagent, and react at 25-45°C for 10-60 minutes , the nucleic acid amplification can be completed.

The most important feature of the method of the present invention is that it can realize nucleic acid amplification at a temperature lower than that in PCR technology or isothermal nucleic acid amplification technology, and only needs a simple and cheap small-scale constant temperature device, even when the ambient temperature reaches 25°C and In the above cases, the reaction can be completed directly by placing it at room temperature without using a traditional PCR instrument, and the operation is extremely simple. the

In addition, because the E. coli helicase RecQ protein, UvsY protein, and UvsX protein are highly specific to the target sequence during the amplification process, in the reaction system of complex genomic DNA, only the position where the primer and the template sequence are completely complementary will be Amplification occurs, thereby avoiding the non-specific background common in conventional PCR reactions (conventional PCR can still cause non-specific amplification when there are several bases in the middle of the primer that are not complementary), so the specificity of the amplification result can be improved Greatly improved, so as to achieve high-efficiency isothermal nucleic acid amplification without background.

Compared with traditional PCR technology, this technology is more suitable for the detection of a large number of samples, almost no professional equipment is needed, the operation is simpler, the technical requirements for operators are lower, and the reaction time is only about half an hour. At the same time, the present invention can also use nucleic acid fluorescent dyes visible to the naked eye to judge preliminary results. If a positive result occurs, gel electrophoresis can be performed to further identify the amplified nucleic acid molecules.

Description of drawings

Fig. 1 is the product gel electrophoresis pattern after the transgenic tobacco chloroplast DNA of FMDV antigen VP1 gene is amplified by PCR and isothermal nucleic acid amplification method of the present invention;

Fig. 2 is the product gel electrophoresis pattern after the transgenic tobacco genomic DNA of superoxide dismutase (Mn-SOD) gene is amplified by PCR and isothermal nucleic acid amplification method of the present invention;

Wherein, M is a marker, 1 is a PCR amplification product, and 2 is an amplification product of the isothermal nucleic acid amplification method of the present invention.

Detailed ways

The present invention is described in detail below in conjunction with embodiment.

Example 1 Detection of foot-and-mouth disease virus (FMDV) antigen VP1 gene transgenic tobacco

First, the FMDV antigen VP1 gene is inserted into the vector p16APT containing the tobacco chloroplast-specific Prrn promoter and the psbA terminator to form the expression cassette of the target gene; then the complete expression cassette is excised by restriction enzymes and Inserted into the vector pTRV containing homologous fragments rpl2-trnH-psbA and trnK-ORF509A of tobacco chloroplast genome to form a complete plasmid expressed by tobacco chloroplast containing FMDV antigen VPI gene.

On the other hand, to cultivate sterile tobacco (Nicotiana tabacum), take aseptic tobacco seedlings at the 4-6 leaf stage, select the expanded leaves of tobacco sterile seedlings with a diameter of about 3 cm as explants, and place them in a 9 cm sterile culture dish center. There is a thin layer of MS medium containing 0.2mol/L mannitol and 0.2mol/L sorbitol in the petri dish, which can be buffered for bombardment. Using the gene gun transformation method, the constructed plasmid was transferred to the chloroplast of tobacco, and the transformed strain of tobacco was obtained by screening with antibiotics (spectinomycin) (see 2003, Zhejiang University, Foot-and-mouth disease virus VP1 antigen gene in chloroplast of model plant Recombination and expression in ).

Tobacco chloroplast DNA extraction (high ionic strength low pH method):

After washing 50g of tobacco leaves with distilled water and drying them, place them at 4°C and starve overnight in the dark;

Remove the veins, chop the leaves and put them in a blender;

Add 200mL 4℃ pre-cooled buffer A, homogenize several times at high speed, 10sec each time, until it becomes a paste;

Buffer A: 25mmol/L Citrate, 1.25mol/L NaCl, 0.25mol/L Vc, pH3.6

Use 8 layers of medical gauze to filter the filtrate into a pre-cooled 250mL centrifuge cup;

Centrifuge at 800g at 4°C for 6min, discard the supernatant;

Add 100mL pre-cooled buffer B to resuspend chloroplast;

buffer B: 50mmol/L Tris-HCl, 25mmol/L EDTA, 1.25mol/L NaCl, 10mmol/L b -mercaptoethanol (pH8.0)

Centrifuge at 800g for 6 minutes at 4°C, discard the supernatant, and recover chloroplast particles;

Add 30mL buffer C to resuspend the pellet;

buffer C: 150mmol/L NaCl, 100mmol/L EDTA(pH8.0)

Centrifuge at 1000g for 8 minutes at 4°C, discard the supernatant, and recover chloroplasts;

Add 8mL buffer D to lightly suspend the precipitate, then add 2mL 10% Sarkosyl (prepared with buffer D), 100mL Protease K (10mg/mL), and shake gently at 40°C for 4-6h;

Buffer D: 50mmol/L Tris-HCl, 25mmol/L EDTA (pH8.0)

Extract with an equal volume of TE saturated phenol (pH8.0) several times, and then extract with phenol:chloroform:isoamyl alcohol (25:24:1) for 1-2 times until the interface is clean;

Add 1/2 volume of 7.5mol/L NH ₄ AC and 2 volumes of absolute ethanol to the supernatant, and place at -20°C for more than 2 hours;

Centrifuge at 12000g for 15min at 4°C to recover DNA, dissolve it in 500mL TE buffer, and transfer it to a 1.5mL eppendorf tube;

Add 5mL RNaseA (10mg/mL), incubate at 37°C for 1h;

Extract with phenol and phenol/chloroform until the interface is clean;

Add 1/10 volume of 3mol/L NaAC (pH5.2) and 2 times the volume of absolute ethanol to the supernatant, rotate horizontally and mix well;

After the white flocculent precipitate appeared, the DNA was recovered by centrifugation at 15000g for 20min at 4°C, dried in vacuum, and dissolved in 100mL TE buffer.

Two methods are used to detect the FMDV antigen VP1 gene, one is a conventional PCR method, and the other is an isothermal nucleic acid amplification method of the present invention.

1. Traditional PCR nucleic acid amplification to prepare the transgenic tobacco positive genome of the FMDV antigen VP1 gene as a positive control

The following primers were designed and commissioned to be synthesized by Beijing Sanbo Yuanzhi Biotechnology Co., Ltd.:

5′ GCATGACCACCTCTGCGGGCG 3′ SEQ ID NO: 1

5′ GCTTACAGAAGCTGTTTTGCG 3′ SEQ ID NO: 2

The selected template sequence is shown in SEQ ID NO: 3.

The PCR system is as follows:

10×PCR buffer 5μL

4×dNTP (10mM each) 1μL

Primer P1 (10μM) (SEQ ID NO: 1) 1μL

Primer P2 (10μM) (SEQ ID NO: 2) 1μL

Template DNA 1 μL

Taq enzyme (Takara company) 1 μL

ddH ₂ O 41μL

The total volume is 50 μL.

Add the above ingredients to a 0.5ml eppendrof tube, put it into the PCR machine,

Reaction procedure:

Pre-denaturation at 95°C for 5 minutes;

94°C for 1 min, 57°C for 1 min, 72°C for 2 min, 28 cycles;

Extend at 72°C for 10 minutes.

After the reaction was completed, the amplified products were identified by electrophoresis.

2. Isothermal nucleic acid amplification

The following primers were designed and commissioned to be synthesized by Beijing Sanbo Yuanzhi Biotechnology Co., Ltd.:

5′ GGATGACCACCTCTGCGGGCGAGTCCGCGG 3′ SEQ ID NO: 4

5′ GCTTACAGAAGCTGTTTTGCGGGTGCCACG 3′ SEQ ID NO: 5

The proportioning of nucleic acid amplification reaction reagents is as follows:

Tris(PH 7.4) 50mM

Potassium acetate 50 mM

Magnesium acetate 10 mM

Dithiothreitol 5 mM

PEG (15000-20000) 10% (w/v)

ATP 10 mM

dNTPs 2 mM

Creatine kinase 2.7μg/U

Phosphocreatine 50 mM

Primer A (SEQ ID NO: 4) 10 μM

Primer B (SEQ ID NO:5) 10 μM

SSB protein 800 ng/μL

RecQ protein 10 ng/μL

UvsX protein 60 ng/μL

UvsY protein 50 ng/μL

Recombinant Klenow exo-polymerase 50 ng/μL

SYTO-13 0.5mM.       

Take 2 μL of the extracted tobacco chloroplast DNA and add it to an eppendrof tube, then add 25 μL of the above nucleic acid amplification reagent, keep it warm at 25°C for 1 hour, take out the liquid in the observation tube and the color is green.

The amplified product was further identified by electrophoresis, and a 645bp band appeared as a result, as shown in Figure 1 .

The detection of embodiment 2 Mn-SOD gene transgenic tobacco

In this example, the superoxide dismutase (Mn-SOD) gene was first inserted into the vector pRTL2 containing the tobacco cell nucleus E35S promoter, TEV Leader and 35S terminator to form the expression cassette of the target gene. Then the complete expression cassette is inserted into the tobacco transformation vector Pzp211 through the action of the restriction endonuclease pstI to form a complete plasmid expressing in the tobacco cell nucleus containing the superoxide dismutase (Mn-SOD) gene.

On the other hand, cultivating aseptic tobacco seedlings: Sow the seeds of tobacco leaves on MS medium under aseptic conditions, and take the young leaves of the aseptic seedlings for transformation after about 6-8 weeks. Tobacco leaf discs were infected with Agrobacterium containing the recombinant plasmid. Sterile tobacco leaf discs need to be pre-cultured on MS1 solid medium for 3 days. After the Agrobacterium was cultivated to the logarithmic phase, it was diluted with liquid MS medium to form an infection solution. Screening of resistant regenerated plants: adding a certain concentration of appropriate antibiotics to the culture medium to screen the regenerated plants to obtain complete transgenic regenerated plants with resistance.

CTAB method for extracting tobacco genomic DNA:

Weigh 0.1 g-0.2 g leaves, add liquid nitrogen into a 1.5 mL centrifuge tube, and quickly grind into light yellow powder with a glass rod;

Add 600 μL of 2% CTAB buffer and 5 μL of RNaseA (10 mg/mL), mix well and incubate at 65°C for 30 min, during which time, gently invert and mix once every 10 min;

Add an equal volume of phenol-chloroform (v/v = 1:1) and mix well, let stand for 3 min, centrifuge at 10,000 rpm for 10 min at 4°C;

After centrifugation, pipette the supernatant into a new 1.5 mL centrifuge tube, add an equal volume of chloroform, mix and let stand for 5 min, centrifuge at 10,000 rpm, 4°C for 10 min;

After centrifugation, draw the supernatant into a new 1.5 mL centrifuge tube, add 2/3 of the volume of isopropanol, gently invert and mix well, so that the isopropanol and the water layer are fully mixed until DNA flocs can be seen, and then set Precipitate in -20℃ refrigerator for 30 minutes;

Take out the sample, centrifuge at 12000 rpm at 4°C for 3 min, pour off the supernatant gently, and be careful not to pour out the white DNA precipitate;

Add 1.0 mL of 70% pre-cooled absolute ethanol, centrifuge at 12000 rpm at 4°C for 2 min, discard the supernatant, and repeat 3 times;

Centrifuge quickly and carefully absorb the liquid from the bottom of the centrifuge tube;

After drying in a 37°C incubator, 50 μL TE buffer was added to dissolve the DNA, and finally placed in a -20°C refrigerator for later use.

In this experiment, two methods were used to detect the obtained transformed strains, one was the conventional PCR method, and the other was the isothermal nucleic acid amplification method of the present invention. The primers in this example were all designed by the applicant and commissioned to be synthesized by Beijing Sanbo Yuanzhi Biotechnology Co., Ltd.

1. Traditional PCR nucleic acid amplification to prepare transgenic tobacco positive genome of superoxide dismutase (Mn-SOD) gene as positive control

The following primers were designed and synthesized:

5′ GCATGCCATTTGAATTGCCAG 3′ SEQ ID NO: 6

5′ GCTTACTTCGCTTTCGCTTCG 3′ SEQ ID NO: 7

The selected template sequence is shown in SEQ ID NO: 8.

The PCR amplification system is as follows:

_ddHO : 19.5 μL

10×buffer: 2.5 μL

dNTP (each 10mM): 0.5 μL

Genomic DNA: 1 μL

Upstream primer (10mM) (SEQ ID NO: 6): 0.5 μL

Downstream primer (10mM) (SEQ ID NO: 7): 0.5 μL

Taq enzyme (Takara): 0.5 μL

 

 

Total volume: 25 μL.

Add the above components into a 0.5ml eppendrof tube and put it into the PCR instrument. The procedure is as follows: (The annealing temperature and extension time can be adjusted according to the Tm value of the primer and the length of the amplicon)

Pre-denaturation: 95 ℃ for 5 min

Denaturation: 95 ℃ 30 s

Annealing: 56 ℃ 60 s

Extension: 72 ℃ 50 s

Number of cycles: 30 cycles

Extension: 72 ℃ for 10 min.

After the reaction was completed, the amplified products were identified by electrophoresis.

2. Isothermal nucleic acid amplification

The following primers were designed and synthesized:

5′ GCATGCCATTTGAATTGCCAGCATTGCCGTATC 3′ SEQ ID No: 9

5′ GCTTACTTCGCTTTCGCTTCGCTGTACCGTTTC 3′ SEQ ID No: 10

The proportioning of nucleic acid amplification reaction reagents is as follows:

Tris(PH 7.4) 50mM

Potassium acetate 50 mM

Magnesium acetate 10 mM

Dithiothreitol 5 mM

PEG (15000-20000) 10% (w/v)

ATP 10 mM

dNTPs 2 mM

Creatine kinase 2.7μg/U

Creatine Phosphate 50 mM

Primer A (SEQ ID NO: 9) 10 μM

Primer B (SEQ ID NO: 10) 10 μM

SSB protein 800 ng/μL

RecQ protein 10 ng/μL

UvsX protein 60 ng/μL

UvsY protein 50 ng/μL

Recombinant Klenow exo-polymerase 50 ng/μL

SYTO-13 0.5mM. the

Take 2 μL of the extracted tobacco genomic DNA and add it to an eppendrof tube, then add 25 μL of the above nucleic acid amplification reagent, incubate at 37°C for 1 hour, take out and observe that the liquid in the tube is green.

The amplified product was further identified by electrophoresis, and a 615bp band appeared as a result, as shown in Figure 2.

The above examples illustrate that the nucleic acid amplification method using the nucleic acid amplification reagent of the present invention can effectively detect the target gene. Compared with the conventional PCR nucleic acid amplification technology, it is easy to operate, the time used is greatly reduced, and no large-scale equipment is required. It is applicable to Large-scale screening.

sequence listing

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Claims (7)

1. An isothermal nucleic acid amplification reaction reagent, characterized in that it comprises the following components:

Wherein, the molecular weight of the polyethylene glycol is 15000-20000. 2. a kind of isothermal nucleic acid amplification reaction reagent as claimed in claim 1, is characterized in that: described DNA polymerase is recombinant Klewnow exo-polymerase. 3. The isothermal nucleic acid amplification reaction reagent according to claim 1, characterized in that: the pH value of the Tris solution is 7.4. 4. The reaction reagent for isothermal nucleic acid amplification according to claim 1, wherein the reaction reagent further comprises a fluorescent dye. 5 . The isothermal nucleic acid amplification reaction reagent according to claim 4 , wherein the fluorescent dye is SYBRgreen I, SYTO-13 or SYTO-82. 6. A method for isothermal nucleic acid amplification, characterized in that: comprising the steps of: 1. extracting DNA, or reverse transcription after extracting RNA to obtain cDNA; 2. adding the isothermal nucleic acid as described in any one of claims 1-5 The amplification reaction reagent can be reacted at 25-45°C for 10-60 minutes to complete the nucleic acid amplification. 7. The isothermal nucleic acid amplification method according to claim 6, wherein the temperature of the nucleic acid amplification reaction is 37°C.

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