CN117343999B - Nucleic acid amplification method based on left-side probe annealing and right-side probe annealing extension - Google Patents
Nucleic acid amplification method based on left-side probe annealing and right-side probe annealing extension Download PDFInfo
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention discloses a nucleic acid amplification method based on left-side probe annealing and right-side probe annealing extension, which is characterized by comprising the following steps: s1, preparing a left free probe, a right free probe and a template nucleic acid; s2, generating an annealed left probe Lp in a test tube L, and immediately performing an ice-water bath; s3, generating an annealed right-side probe Rp in a test tube R, performing primary extension to generate an annealed extended right-side probe Rp', and immediately performing an ice-water bath; s4, sorting and purifying the test tube L and the test tube R; s5, performing PCR amplification on the products after sorting and purifying the test tube L and the test tube R in the step S4. The nucleic acid amplification method based on the left side probe annealing and the right side probe annealing extension disclosed by the invention has the advantages of high concentration of amplified nucleic acid products, simple steps, low experiment cost and short time consumption, and is suitable for detecting the nucleic acid sequences among probes.
Description
Technical Field
The invention relates to the field of nucleic acid amplification, in particular to a nucleic acid amplification method based on left-side probe annealing and right-side probe annealing extension.
Background
In the detection of samples, the target nucleic acid of interest sometimes cannot be detected efficiently due to its very low content compared to the background nucleic acid [1], so that it is necessary to perform appropriate enrichment/amplification of the target nucleic acid in advance [2]. Strategies directed to enrichment/amplification of target nucleic acid fragments in combination with nucleic acid detection have a wide range of applications, such as for monitoring and diagnosis of infectious diseases [3, 4], sequencing of viral genomes in patient samples [5], genotyping of pathogenic microorganisms [4], tumor diagnosis and prognosis [6, 7], etc.
For nucleic acid detection, common methods include (capillary) nucleic acid electrophoresis [8], sanger sequencing/second generation sequencing (next-generation sequencing, NGS)/third generation sequencing (third-generation sequencing) [9,10], CRISPR/Cas [11] based, use of fluorescent probes/nucleic acid dyes [12, 13], nucleic acid mass spectrometry [7], indication of pH differences before and after PCR [14], and the like.
For enrichment/amplification of target nucleic acid fragments, common methods include (multiplex) PCR [15], isothermal amplification [16], CRISPR/Cas [2], differential cleavage enrichment of pathogenic microorganism nucleic acids [17], solid phase chip hybridization capture (microarray hybridization) [18], liquid phase hybridization capture (in solution hybridization) [19], molecular inversion probe (molecular inversion prob, MIP) capture [20, 21], multiplex ligation dependent probe amplification (multiplex ligation-dependent probe amplification, MLPA) [8], and the like. Common methods in the prior art, such as solid-phase chip hybridization capture, liquid-phase hybridization capture, MIP capture and MLPA, belong to targeted enrichment/amplification methods, and the flux of the enriched/amplified target nucleic acid has expandability, but still has certain drawbacks: the hybrid capture method has high performance, but is still expensive and time consuming, and requires large amounts of nucleic acid [8, 22]; the MIP capturing method uses longer probes, which consist of a middle general interval sequence and target nucleic acid specific sequences at two sides, and can affect the capturing efficiency of the target nucleic acid, and in addition, the MIP capturing method also comprises DNA connection and exonuclease digestion steps, and the steps are more complicated [13, 14]; the target nucleic acid specific flanking probes used in MLPAs are adjacent (i.e., no nucleotide gaps) at the annealing site of the target nucleic acid, which is annealed to the target nucleic acid simultaneously and ligated by a ligase, and thus are not suitable for detecting nucleic acid sequences between probes [8, 23].
Disclosure of Invention
The object of the present invention is to provide a nucleic acid amplification method based on left-side probe annealing and right-side probe annealing extension.
The nucleic acid amplification method based on the left side probe annealing and the right side probe annealing extension provided by the invention comprises the following steps:
s1, preparing a left free probe with a universal primer binding site, a right free probe with a universal primer binding site and a template nucleic acid;
s2, performing an annealing procedure on the left free probe and the template nucleic acid in a test tube L to generate an annealed left probe Lp (left probe), and immediately performing an ice-water bath;
s3, performing an annealing procedure on the right free probe and the template nucleic acid in a test tube R to generate an annealed right probe Rp (right probe), and performing primary extension by using a DNA polymerase with 3' - >5' exonuclease activity to generate an annealed extended right probe Rp ', and immediately performing an ice-water bath;
s4, sorting and purifying the test tube L and the test tube R by using DNA sorting magnetic beads respectively;
s5, performing PCR amplification on the products after sorting and purifying the test tube L and the test tube R in the step S4.
Further, the step S2 for annealing the left free probe and the template nucleic acid in the test tube L to generate an annealed left probe Lp, and immediately performing an ice-water bath, comprises:
s21 is used for carrying out high-temperature denaturation on the test tube L at 90-100 ℃ for 3-10 minutes, preferably 98 ℃ for 3 minutes;
s22 is used for carrying out low-temperature annealing on the test tube L after high-temperature denaturation, wherein the temperature is 50-72 ℃, the time is 1-60 minutes, preferably 65 ℃ and the time is 5 minutes;
s23 is used for carrying out ice-water bath on the test tube L after low-temperature annealing for 1-60 minutes, preferably 5 minutes.
Further, the step S3 for annealing the right free probe and the template nucleic acid in the test tube R to generate an annealed right probe Rp, and performing one-time extension with a DNA polymerase having 3' - >5' exonuclease activity to generate an annealed extended right probe Rp ', immediately after which an ice-water bath is performed, comprises:
s31 is used for carrying out high-temperature denaturation on the test tube R, wherein the temperature is 90-100 ℃, the time is 3-10 minutes, preferably 98 ℃, and the time is 3 minutes;
s32 is used for carrying out low-temperature annealing on the test tube R after high-temperature denaturation, wherein the temperature is 50-72 ℃, the time is 1-60 minutes, preferably 65 ℃, and the time is 5 minutes;
s33 is used for performing primary extension in the test tube R subjected to low-temperature annealing by using DNA polymerase with 3'- >5' exonuclease activity, wherein the temperature is kept at 65-72 ℃ for 5-60 seconds, preferably 72 ℃ for 10 seconds;
s34 is used to subject the extended tube R to an ice-water bath for a period of 1-60 minutes, preferably 5 minutes.
Further, the step S3 is used for annealing the free right probe and the template nucleic acid in the test tube R to generate an annealed right probe Rp, and performing one-time extension with a DNA polymerase having 3' - >5' exonuclease activity to generate an annealed extended right probe Rp ', and immediately after that, the step further comprises the steps of:
s35 is used for adding proteinase K into the test tube R in the step S34 to degrade the DNA polymerase, wherein the temperature is 37-65 ℃ for 5-60 minutes, preferably 65 ℃ for 30 minutes;
s36 is used for carrying out high-temperature denaturation on the test tube R in the step S35, wherein the temperature is 90-100 ℃, the time is 3-10 minutes, preferably 98 ℃, and the time is 10 minutes;
s37 is used for carrying out low-temperature annealing on the test tube R in the step S36 at 50-72 ℃ for 1-60 minutes
Clock, preferably 65 degrees celsius, for 5 minutes;
s38 is used to subject the tube R in step S37 to an ice-water bath for a period of 1-60 minutes, preferably 5 minutes.
Further, the S4 is used for separating and purifying the test tube L and the test tube R by using DNA separating magnetic beads respectively,
comprising the following steps:
s41, adding a proper amount of magnetic beads into the test tube L in the step S23 and the test tube R in the step S38 respectively, and carrying out sorting purification by adopting a round method.
Further, the step S5 is used for performing PCR amplification on the products after sorting and purifying the test tube L and the test tube R in the step S4, and includes:
s51 is used for preferential annealing and extension of trace Lp in excess of the upstream primer F and the template nucleic acid to produce trace F';
s52 is used for F ' and Rp ' annealing and F ' extension occurs, using a DNA polymerase with 3' - >5' exonuclease activity;
s53 is used for normal PCR amplification of the upstream primer F and the downstream primer R by taking the product obtained in the step S52 as a template.
Compared with the traditional amplification method, the nucleic acid amplification method based on the left probe annealing and the right probe annealing extension provided by the invention has the advantages that:
1. compared with a hybridization capturing method, the method has the advantages of high performance, small demand for nucleic acid, short test time and low test cost;
2. compared with the MIP capture method, the probe used in the method is short, and the capture efficiency of the target nucleic acid is not affected;
3. the present application is applicable to detecting nucleic acid sequences between probes, as compared to the MLPA capture method.
Drawings
FIG. 1 is a schematic diagram showing steps of a nucleic acid amplification method based on left-side probe annealing and right-side probe annealing extension according to the present invention;
FIG. 2 is a schematic diagram showing the structure and annealing positions of the full-length EGFP and the left and right probes according to example 1 of the present invention;
FIG. 3 is a schematic diagram showing PCR amplification comparison of the right free probe of example 1 of the present invention after annealing and one extension (Tube R) and annealing only (Tube R');
FIG. 4 is a graph showing the contrast ratio of the final PCR product gel mediated by tube R and tube R' according to example 1 of the present invention;
FIG. 5 is a schematic flow chart of the PCR product reduction caused by the test tube R' according to the embodiment 1 of the present invention;
fig. 6 is a diagram of an electrophoresis gel according to embodiment 2 and embodiment 3 of the present invention;
FIG. 7 is a schematic representation of the positions of GFP1, GFP2 and GFP3 fragments of EGFP of example 4 of the present invention;
FIG. 8 is a diagram of electrophoresis gel after single and simultaneous amplification of GFP1, GFP2 and GFP3 in example 4 according to the present invention;
FIG. 9 is a graph showing the sequencing result of the larger amplification product of example 5 of the present invention;
FIG. 10 is a schematic diagram showing the cause of the generation of the larger amplification product of example 5 of the present invention;
FIG. 11 is a diagram of the electrophoresis gel of the PCR products before and after magnetic bead purification in example 6 of the present invention;
FIG. 12 is a graph showing the comparison of the differences in components of the template system before and after magnetic bead purification in example 6 of the present invention;
FIG. 13 is a diagram of an electrophoresis gel for inhibiting PCR amplification by a free probe according to example 6 of the present invention;
FIG. 14 is a diagram of an electrophoresis gel of the individual and simultaneous amplification of human Cg313, cg540, cg929 fragments according to example 7 of the present invention;
FIG. 15 is a schematic diagram showing the structure of amplified PCR products according to examples 1 to 4, 6 and 7 of the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
As shown in fig. 1 to 15, the nucleic acid amplification method based on the extension of the left-side probe annealing and the right-side probe annealing provided in this embodiment includes the following steps:
s1, preparing a left free probe with a universal primer binding site, a right free probe with a universal primer binding site and a template nucleic acid;
s2, performing an annealing procedure on the left free probe and the template nucleic acid in a test tube L to generate an annealed left probe Lp (left probe), and immediately performing an ice-water bath;
s3, performing an annealing procedure on the right free probe and the template nucleic acid in a test tube R to generate an annealed right probe Rp (right probe), and performing primary extension by using a DNA polymerase with 3' - >5' exonuclease activity to generate an annealed extended right probe Rp ', and immediately performing an ice-water bath;
s4, sorting and purifying the test tube L and the test tube R by using DNA sorting magnetic beads respectively;
s5, performing PCR amplification on the products after sorting and purifying the test tube L and the test tube R in the step S4.
It will be appreciated by those skilled in the art that the method for amplifying nucleic acid based on the annealing of the left side probe and the annealing extension of the right side probe provided in this embodiment has high concentration of amplified nucleic acid product, simple steps, low experiment cost, short time consumption, and suitability for detecting nucleic acid sequences between probes.
Further, the step S2 for annealing the left free probe and the template nucleic acid in the test tube L to generate an annealed left probe Lp, and immediately performing an ice-water bath, comprises:
s21 is used for carrying out high-temperature denaturation on the test tube L at 90-100 ℃ for 3-10 minutes, preferably 98 ℃ for 3 minutes;
s22 is used for carrying out low-temperature annealing on the test tube L after high-temperature denaturation, wherein the temperature is 50-72 ℃, the time is 1-60 minutes, preferably 65 ℃ and the time is 5 minutes;
s23 is used for carrying out ice-water bath on the test tube L after low-temperature annealing for 1-60 minutes, preferably 5 minutes.
Further, the step S3 for annealing the right free probe and the template nucleic acid in the test tube R to generate an annealed right probe Rp, and performing one-time extension with a DNA polymerase having 3' - >5' exonuclease activity to generate an annealed extended right probe Rp ', immediately after which an ice-water bath is performed, comprises:
s31 is used for carrying out high-temperature denaturation on the test tube R, wherein the temperature is 90-100 ℃, the time is 3-10 minutes, preferably 98 ℃, and the time is 3 minutes;
s32 is used for carrying out low-temperature annealing on the test tube R after high-temperature denaturation, wherein the temperature is 50-72 ℃, the time is 1-60 minutes, preferably 65 ℃, and the time is 5 minutes;
s33 is used for performing primary extension in the test tube R subjected to low-temperature annealing by using DNA polymerase with 3'- >5' exonuclease activity, wherein the temperature is kept at 65-72 ℃ for 5-60 seconds, preferably 72 ℃ for 10 seconds;
s34 is used to subject the extended tube R to an ice-water bath for a period of 1-60 minutes, preferably 5 minutes.
Further, the step S3 is used for annealing the free right probe and the template nucleic acid in the test tube R to generate an annealed right probe Rp, and performing one-time extension with a DNA polymerase having 3' - >5' exonuclease activity to generate an annealed extended right probe Rp ', and immediately after that, the step further comprises the steps of:
s35 is used for adding proteinase K into the test tube R in the step S34 to degrade the DNA polymerase, wherein the temperature is 37-65 ℃ for 5-60 minutes, preferably 65 ℃ for 30 minutes;
s36 is used for carrying out high-temperature denaturation on the test tube R in the step S35, wherein the temperature is 90-100 ℃, the time is 3-10 minutes, preferably 98 ℃, and the time is 10 minutes;
s37 is used for carrying out low-temperature annealing on the test tube R in the step S36, wherein the temperature is 50-72 ℃, the time is 1-60 minutes, preferably 65 ℃ and the time is 5 minutes;
s38 is used to subject the tube R in step S37 to an ice-water bath for a period of 1-60 minutes, preferably 5 minutes.
It will be appreciated by those skilled in the art that proteinase K is inactivated at a high temperature after cleavage.
Further, the S4 is used for separating and purifying the test tube L and the test tube R by using DNA separating magnetic beads respectively,
comprising the following steps:
s41, adding a proper amount of magnetic beads into the test tube L in the step S23 and the test tube R in the step S38 respectively, and carrying out sorting purification by adopting a round method. The purpose of magnetic bead purification is understood by those skilled in the art: recovering the template and Lp annealed thereto or Rp and Rp' annealed/extended thereto; the left and right free probes that were not annealed were removed. Purifying DNA magnetic beads: magnetic beads (next holy, china) were sorted using 1.5 Xvolume Hieff NGS DNA and were subjected to one round of sorting purification according to the purification scheme of the specification.
Further, the step S5 is used for performing PCR amplification on the products after sorting and purifying the test tube L and the test tube R in the step S4, and includes:
s51 is used for preferential annealing and extension of trace Lp in excess of the upstream primer F and the template nucleic acid to produce trace F';
s52 is used for F ' and Rp ' annealing and F ' extension occurs, using a DNA polymerase with 3' - >5' exonuclease activity; as will be appreciated by those skilled in the art, F 'and Rp' annealing and extension occur automatically during the PCR run, and the temperature and time are set by the PCR apparatus using a DNA polymerase with 3'- >5' exonuclease activity.
S53 is used for normal PCR amplification of the upstream primer F and the downstream primer R by taking the product obtained in the step S52 as a template.
It will be appreciated by those skilled in the art that the PCR polymerase used has 3'- >5' exonuclease activity. In the PCR process, compared with the limited template nucleic acid, the excessive upstream primer F is firstly annealed and extended preferentially with trace Lp in the template nucleic acid to generate trace F ', and finally F, F ' and the downstream primer R are subjected to normal PCR amplification by taking Rp ' as templates. Flanking sequences can be introduced at the 5' ends of F and R according to the actual requirements which follow. And (3) PCR amplification: the polymerase used for PCR was Q5 Hot Start High-fidelity DNA polymerase (Q5 Hot Start High-fidelity DNA Polymerase, Q5 polymerase for short) (NEB, USA), and the reaction system and amplification were configured according to the instructions. The upstream primer F and the downstream primer R are both of the prior art, and are not described in detail herein.
Further, the template nucleic acid used in example 1, example 3 to example 6 was a plasmid containing the full length EGFP, namely pcDNA3.1 (+) -EGFP. The template nucleic acid used in example 2 was a plasmid containing GFP1, namely pcDNA3.1 (+) -GFP1. The template of examples 1-6 was used in an amount of 50 ng. The relevant nucleotide sequences used in examples 1-6 are shown in Table 1.
Table 1: examples 1 to 6 related nucleotide sequences used
Example 1:
after annealing of the tube R and the template nucleic acid and one extension and annealing of the template nucleic acid only (FIGS. 2 and 3), the remaining identical steps (FIG. 1), including PCR, are continued. By comparing the PCR products, after PCR amplification of the annealing program and the tube R (tube) after one extension, the lane of "tube" in the electrophoresis gel chart is significantly brighter than the lane of "tube '" indicating that the concentration of the amplified product in the tube R is significantly higher than that in the tube R ' (tube ') after only the annealing program (FIG. 4).
The reaction systems of tube L, tube R and tube R' are shown in Table 2;
table 2: reaction system of test tube L, test tube R and test tube R
As will be appreciated by those skilled in the art, the reason that tube R' results in a reduction in PCR product is: if the free probe on the right is only annealed to the template nucleic acid and not extended, then during PCR amplification with both purified products as templates after the separate bead purification of tube L and tube R ', rp and Lp may anneal to the same template nucleic acid molecule at the same time because the strand displacement activity of the Q5 polymerase is very low [24], thus most of the annealed Lp blocks extension of Rp compared to Rp', resulting in the extended product not comprising the annealing site corresponding to Lp and eventually in a reduced amplified product (FIG. 5).
Further, the magnetic bead purification is performed to remove the left free probe and the right free probe by magnetic bead purification;
example 2:
after mixing the plasmid pGFP1 and the right probe GFP2Rp (FIG. 7), 1, after purification by magnetic beads, it was found by electrophoresis gel (FIG. 6) that only one band was present in the "+" lane in the gel, indicating that pGFP1 was present in the tube after purification by magnetic beads and GFP2Rp had been removed; 2. without purification by magnetic beads, two bands were still present in the "-" lanes in the gel by electrophoresis gel, indicating that pGFP1 and GFP2Rp were still contained in the tube without purification by magnetic beads and GFP2Rp was not removed.
pGFP1 is a plasmid containing the GFP1 sequence (pcDNA3.1 (+) -GFP 1); GFP2Rp is the right free probe segment corresponding to the GFP2 segment (fig. 7).
Example 3:
annealing procedure for plasmid pEGFP and 3 left free probes (fig. 7) in tube L, annealing procedure and one extension procedure for pEGFP and 3 right free probes (fig. 7) in tube R, followed by mixing tube L and tube R, 1, after purification by magnetic beads, electrophoresis gel shows that only one band is present in the "+" lane, indicating that only pEGFP is present in the tube after mixing; 2. after purification by magnetic beads, the electrophoresis gel shows that the "-" lanes present two bands, indicating that after mixing, the free probe was not removed (FIG. 6).
It will be appreciated by those skilled in the art that examples 2 and 3 above demonstrate that free probes in the mixing tube can be effectively removed by magnetic bead purification.
Example 4:
for the amplification of 3 different segments of EGFP, respectively, the electrophoresis gel diagram shows that the "GFP1", "GFP2", "GFP3"3 lanes all obtain similar amplified bands (FIG. 8);
for simultaneous amplification of 3 different segments of EGFP, the electrophoresis gel diagram shows that there is a bright main band in the "GFP1&2&3" lanes, while also a larger faint amplified band appears (FIG. 8);
example for amplification of 3 different segments of EGFP, 3 of which are three segments of GFP1, GFP2, GFP3 on EGFP (FIG. 7).
Example 5:
PCR product purification and cloning and sequencing were performed on the simultaneously enriched/amplified products, and the results of the examples show that:
of the 18 clones sequenced successfully, 6 clones were specifically aligned to GFP1,4 clones were specifically aligned to GFP2,7 clones were specifically aligned to GFP3, and 1 clone was also simultaneously aligned to GFP2 and GFP3, i.e.the ligation products were larger (FIGS. 8 and 9); the reason for the larger ligation products may be that when GFP3Rp is extended, the Q5DNA polymerase used is due to weak strand displacement activity [24] displacing the downstream GFP2Rp (FIG. 10).
Those skilled in the art will appreciate cloning and sequencing of DNA products: the PCR product amplified by Q5 polymerase was recovered by using MolPure PCR purification kit (Saint Bio, china), and then the reaction system was prepared by using Hieff Taq DNA polymerase (Saint bio, china) and according to the instructions thereof, and was treated at 72℃for 10 minutes to add base A at the 3' -end of DNA. The DNA product with base A added was TA ligated using Hieff Clone zero background TOPO-TA cloning kit (next holy, china). Or the recovered PCR products were directly ligated using Hieff Clone zero background TOPO-Blunt Blunt end cloning kit (Style of the next, china). The ligated products were transformed into E.coli Top10 competent cells, coated with ampicillin LB plates, and the monoclonal selected (without blue-white screening) for incubation and used for sequencing. The primer used for sequencing is a vector universal sequencing primer, namely an M13 reverse sequencing primer.
The free probe inhibited PCR amplification, as demonstrated in example 6 below:
example 6:
"Template: 13.4 μl": 20. mu L of the PCR system is supplemented with templates before or after magnetic bead purification; "Template: 0.5 μl": to 20. Mu.L of the PCR system, 0.5. Mu.L of the template before or after magnetic bead purification was added, and the remaining volume (i.e., 12.9. Mu.L) was filled with water. The nucleic acid templates used are templates corresponding to GFP1, GFP2 and GFP3 before or after magnetic bead purification when amplification is carried out simultaneously (i.e. 3 left free probes and pEGFP annealed, 3 right free probes and pEGFP annealed extended and proteinase K treated); NTC: no template control. It will be appreciated by those skilled in the art that unlike the templates after magnetic bead purification, PCR amplification was inhibited when large volumes of templates before magnetic bead purification were added, and non-specific amplification bands appeared (FIG. 11).
By analysis, it is found that:
before magnetic bead purification, the system contains a large amount of Q5 Reaction Buffer (Q5 Reaction Buffer), dNTPs (deoxyribonucleotides) and free probes (free probes), and after magnetic bead purification, the system does not contain Q5 Reaction Buffer, dNTPs and free probes (figure 12);
in some cases, the target nucleic acid content in the nucleic acid is extremely low, so in order to capture such nucleic acids, it is necessary to add as large a volume (or all) of template as possible in the PCR system. Unlike the template after magnetic bead purification, PCR amplification was inhibited when a large volume of the template before magnetic bead purification was added, and a nonspecific amplification band appeared. Compared with the magnetic bead purification, the template before the magnetic bead purification contains Q5 Reaction Buffer, dNTPs, free probes and Q5DNA polymerase. In order to further clarify the specific components for inhibiting PCR, the high concentration Q5 Reaction Buffer, high concentration dNTPs and high concentration free probes corresponding to the template before large volume magnetic bead purification were simulated during PCR, and the result showed that the high concentration free probes inhibited PCR amplification, and that the nonspecific amplification band was probably derived from the mutual annealing amplification between the upstream primer F and the left free probe, and the result of this example showed that the high concentration Q5 Reaction Buffer also had an effect on the PCR amplification efficiency (FIG. 13), wherein the concentration of FIG. 13 represents concentration.
Further, the template nucleic acid used in this example 7 was genomic DNA of the human cell line 293T (the amount of template used was 150 ng), and the detected target was 3 DNA fragments derived from the human genome. The relevant nucleotide sequences used are shown in Table 3:
table 3: example 7 related nucleotide sequences used
As can be appreciated by those skilled in the art: the 5 phosphodiester bonds at the 3' end introduce thio modifications in order to block the cleavage of the 3' - >5' of the magnetic bead-bound nucleic acid by the Q5 polymerase, which is indirectly bound to the magnetic bead through the nucleic acid during magnetic bead purification.
Amplification experiments were performed on human Cg313, cg540, cg929 fragments, using gDNA extracted from 293T cells, NTC: template-free control:
example 7:
the Cg540, cg313 and Cg929 fragments of the human are respectively amplified and simultaneously amplified, and four lanes of Cg540, cg313, cg929 and Cg540&313&929 are respectively and consistently amplified through an electrophoresis gel diagram (figure 14);
the PCR product was purified and cloned and sequenced from the simultaneously amplified products, 7 clones were specifically aligned to Cg540, 10 clones were specifically aligned to Cg313,9 clones were specifically aligned to Cg929, and the other 4 clones were amplified from each other between primers/probes, among 30 clones sequenced successfully.
The PCR products of examples 1-4, 6, 7 contained additional sequences (FIG. 15), P5, P7 respectively: sequences for sequencing on the illuminea platform; i5, i7: an index sequence; read1, read2: sequencing primer binding sites for read1 and read 2; tsL, tsR: target specific Lp and Rp binding sites. It will be appreciated by those skilled in the art that PCR products contain P5 and P7 sequences for sequencing on the illuminea platform and thus there is a possibility for sequencing on the illuminean NGS platform. Of course, by rationally altering the sequence and structure of the 5' end of the F/R primer, the LRpM-PCR can be integrated with a variety of subsequent nucleic acid detection platforms/techniques, such as with other two/three generation sequencing platforms [25] or CRISPR/Cas detection platforms [20].
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finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (3)
1. A method for amplifying nucleic acid based on left-side probe annealing and right-side probe annealing extension, comprising the steps of:
s1, preparing a left free probe with a universal primer binding site, a right free probe with a universal primer binding site and a template nucleic acid;
s2, performing an annealing procedure on the left free probe and the template nucleic acid in a test tube L to generate an annealed left probe Lp, and immediately performing an ice-water bath;
s3, performing annealing procedure on the free right probe and the template nucleic acid in a test tube R to generate an annealed right probe Rp, performing primary extension by using DNA polymerase with 3' - >5' exonuclease activity to generate an annealed extended right probe Rp ', and immediately performing ice-water bath;
s4, sorting and purifying the test tube L and the test tube R by using DNA sorting magnetic beads respectively;
s5, performing PCR amplification on the products after separation and purification of the test tube L and the test tube R in the step S4;
the step S2 is used for annealing the left free probe and the template nucleic acid in a test tube L to generate an annealed left probe Lp, and comprises the following steps of:
s21 is used for carrying out high-temperature denaturation on the test tube L, wherein the temperature is 90-100 ℃ and the time is 3-10 minutes;
s22, carrying out low-temperature annealing on the test tube L subjected to high-temperature denaturation at 50-72 ℃ for 1-60 minutes;
s23, carrying out ice-water bath on the test tube L subjected to low-temperature annealing for 1-60 minutes;
the step S3 is used for annealing the free right probe and the template nucleic acid in a test tube R to generate an annealed right probe Rp, and performing one-time extension by using a DNA polymerase with 3' - >5' exonuclease activity to generate an annealed extended right probe Rp ', and immediately after that, performing an ice-water bath, and comprises the following steps:
s31 is used for carrying out high-temperature denaturation on the test tube R, wherein the temperature is 90-100 ℃ and the time is 3-10 minutes;
s32, carrying out low-temperature annealing on the test tube R after high-temperature denaturation, wherein the temperature is 50-72 ℃ and the time is 1-60 minutes;
s33, performing primary extension by using DNA polymerase with 3'- >5' exonuclease activity in a test tube R after low-temperature annealing, wherein the temperature is kept at 65-72 ℃ for 5-60 seconds;
s34, carrying out ice-water bath on the extended test tube R for 1-60 minutes;
the step S5 is used for carrying out PCR amplification on the products after sorting and purifying the test tube L and the test tube R in the step S4, and comprises the following steps:
s51 is used for preferential annealing and extension of the trace left probe Lp in the excess upstream primer F and the template nucleic acid to produce trace F';
s52 is used for F ' and Rp ' annealing and F ' extension occurs, using a DNA polymerase with 3' - >5' exonuclease activity;
s53 is used for normal PCR amplification of the upstream primer F and the downstream primer R by taking the product obtained in the step S52 as a template.
2. The method for amplifying nucleic acid based on annealing of left side probe and annealing extension of right side probe according to claim 1, wherein S3 is a step for annealing the right side free probe and the template nucleic acid in a test tube R to generate annealed right side probe Rp, and extending once with a DNA polymerase having 3' - >5' exonuclease activity to generate annealed extended right side probe Rp ', immediately after which an ice-water bath is further comprised:
s35, adding proteinase K into the test tube R in the step S34 to degrade DNA polymerase, wherein the temperature is 37-65 ℃ and the time is 5-60 minutes;
s36, carrying out high-temperature denaturation on the test tube R in the step S35, wherein the temperature is 90-100 ℃ and the time is 3-10 minutes;
s37, performing low-temperature annealing on the test tube R in the step S36, wherein the temperature is 50-72 ℃ and the time is 1-60 minutes;
s38 is used for carrying out ice-water bath on the test tube R in the step S37 for 1-60 minutes.
3. The method for amplifying nucleic acid based on left-side probe annealing and right-side probe annealing extension according to claim 2, wherein S4 is a step for sorting and purifying test tubes L and R with DNA sorting magnetic beads, respectively,
comprising the following steps:
s41, adding a proper amount of magnetic beads into the test tube L in the step S23 and the test tube R in the step S38 respectively, and carrying out sorting purification by adopting a round method.
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| CN107250361A (en) * | 2015-01-30 | 2017-10-13 | 艾思蒂生物传感器有限公司 | PCR primer and utilize its nucleic acid amplification method that complementary base sequence with complementary base sequence or comprising base mismatch is connected |
| CN112041459A (en) * | 2018-01-29 | 2020-12-04 | 圣祖德儿童研究医院 | Nucleic acid amplification method |
| CN113832258A (en) * | 2020-06-08 | 2021-12-24 | 安普里怀斯公司 | Ribonucleic acid amplification and detection using attenuated probes |
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| CN103354840A (en) * | 2011-01-31 | 2013-10-16 | 欧凌科公司 | Exonuclease enabled proximity extension assays |
| CN107250361A (en) * | 2015-01-30 | 2017-10-13 | 艾思蒂生物传感器有限公司 | PCR primer and utilize its nucleic acid amplification method that complementary base sequence with complementary base sequence or comprising base mismatch is connected |
| CN112041459A (en) * | 2018-01-29 | 2020-12-04 | 圣祖德儿童研究医院 | Nucleic acid amplification method |
| CN113832258A (en) * | 2020-06-08 | 2021-12-24 | 安普里怀斯公司 | Ribonucleic acid amplification and detection using attenuated probes |
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