CN116875671A - Tagged hairpin primers for DNA amplification and labeling - Google Patents
Tagged hairpin primers for DNA amplification and labeling Download PDFInfo
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- 238000002372 labelling Methods 0.000 title claims abstract description 18
- 230000004544 DNA amplification Effects 0.000 title claims abstract description 10
- 238000011901 isothermal amplification Methods 0.000 claims abstract description 43
- 241000700605 Viruses Species 0.000 claims abstract description 13
- 241000203069 Archaea Species 0.000 claims abstract description 4
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- 230000007613 environmental effect Effects 0.000 claims abstract description 4
- 244000005700 microbiome Species 0.000 claims abstract description 4
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 18
- 239000012498 ultrapure water Substances 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 15
- 238000006243 chemical reaction Methods 0.000 claims description 10
- 230000003321 amplification Effects 0.000 claims description 6
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 6
- 239000011535 reaction buffer Substances 0.000 claims description 6
- 238000012163 sequencing technique Methods 0.000 claims description 5
- 210000000601 blood cell Anatomy 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 2
- 108020004414 DNA Proteins 0.000 abstract description 51
- 239000002245 particle Substances 0.000 abstract description 13
- 230000003612 virological effect Effects 0.000 abstract description 10
- 108091028043 Nucleic acid sequence Proteins 0.000 abstract description 7
- 102000053602 DNA Human genes 0.000 abstract 7
- 210000004369 blood Anatomy 0.000 abstract 1
- 239000008280 blood Substances 0.000 abstract 1
- 241000696962 White spot syndrome virus Species 0.000 description 27
- 238000003752 polymerase chain reaction Methods 0.000 description 17
- 108020004707 nucleic acids Proteins 0.000 description 16
- 150000007523 nucleic acids Chemical class 0.000 description 16
- 102000039446 nucleic acids Human genes 0.000 description 16
- 238000000137 annealing Methods 0.000 description 7
- 238000012795 verification Methods 0.000 description 5
- 239000013642 negative control Substances 0.000 description 4
- 108010067770 Endopeptidase K Proteins 0.000 description 3
- 108020005202 Viral DNA Proteins 0.000 description 3
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 241000238557 Decapoda Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
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- 238000012986 modification Methods 0.000 description 1
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- 238000010200 validation analysis Methods 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
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Abstract
The invention discloses a labeled hairpin primer for DNA amplification and labeling, which comprises a DNA (deoxyribonucleic acid) label sequence with 2 or more hairpin structures at the 5' end and a 6-base random primer, wherein the hairpin structures and the 6-base random primer can be directly connected or can be connected through bases; the tag hairpin primer can be used for isothermal amplification of a low-concentration DNA template with unknown or known sequence, wherein the DNA template is derived from environmental DNA, tissue cells of human, animals and plants, blood, body fluid, viruses, bacteria, archaebacteria and the like in microorganisms, and for example, single viral genome DNA can be amplified and labeled through isothermal amplification, so that the whole genome sequence of single viral particles is obtained. The label hairpin primer solves the problem that a sequence specific primer or a random primer in the prior art cannot label an amplified product by using a DNA sequence label while amplifying DNA.
Description
Technical Field
The invention belongs to the technical field of molecular diagnosis, and particularly relates to a labeled hairpin primer for DNA amplification and labeling.
Background
For the amplification of DNA, the prior art adopts a sequence specific primer or a random primer to carry out Polymerase Chain Reaction (PCR) or isothermal amplification, and no technology for marking an amplified product by using a DNA sequence tag while amplifying DNA has been disclosed.
The PCR is performed by adopting a sequence specific primer or a random primer, and the method is mainly used for amplifying the DNA with higher DNA template content, and the isothermal amplification is performed by adopting the sequence specific primer or the random primer, so that the method is suitable for amplifying the DNA with lower DNA template content; if the DNA template sequence to be amplified is known, the sequence-specific primers are used for amplification (the sequence-specific primers are typically linear in structure); if the DNA template sequence to be amplified is unknown, random primers can be used for amplification (the primers are linear structures).
In the above prior art, the sequence-specific primers or random primers employed cannot label the amplified product with a DNA sequence tag at the same time as DNA amplification because:
1) After a section of DNA sequence label is added to the 5' end of the linear structure sequence specific primer, the combination of the sequence specific primer and the template DNA is affected, so that PCR or isothermal amplification cannot be performed, and meanwhile, the label cannot be performed, so that the full-length sequence sequencing of the template DNA cannot be completed.
2) After a section of unknown DNA sequence label is added to the 5' end of the random primer with a linear structure, the random primer cannot be combined with the template DNA, so that PCR or isothermal amplification cannot be performed, meanwhile, the random primer cannot be labeled, and thus, the full-length sequence sequencing of the template DNA cannot be completed.
Disclosure of Invention
Aiming at the technical problems in the background art, the invention aims to provide a labeled hairpin primer for amplifying and labeling DNA, which can simultaneously amplify and label template DNA, and can be used for isothermal amplification of template DNA with unknown or known sequence and low concentration so as to obtain the full-length sequence of the template DNA. Solves the problem that the sequence specific primer or the random primer in the prior art can not be used for marking the amplified product by using a DNA sequence tag while amplifying DNA.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the first aspect of the present invention provides a tagged hairpin primer for DNA amplification and labelling, comprising a DNA tag sequence having 2 or more hairpin structures at the 5' end and a 6 base random primer.
Preferably, the hairpin structure in the tagged hairpin primer is directly linked to a 6 base random primer.
Preferably, the hairpin structure in the tagged hairpin primer is connected with the 6-base random primer through bases, and the number of the bases can be but is not limited to 2 or 4.
In a second aspect, the present invention provides a method for amplifying and labelling DNA using the tagged hairpin primer described above, comprising the steps of:
s1, extracting DNA from a sample to be detected, and taking the DNA as template DNA;
s2, carrying out isothermal amplification reaction on the template DNA by using the labeled hairpin primer and an isothermal amplification reaction system to obtain a labeled isothermal amplification product.
Preferably, in step S2, the composition of the isothermal amplification reaction system includes: template DNA, phi29 polymerase, reaction buffer, dntps, tagged hairpin primers, and nucleotide-free ultrapure water.
Preferably, the isothermal amplification reaction procedure is: 30 ℃ for 3-6h;65 ℃ for 10min; preserving heat at 4 ℃.
Preferably, the sample to be tested is derived from environmental DNA, human and animal tissue cells, blood, body fluids, or viruses, bacteria, archaebacteria, etc. in microorganisms.
A third aspect of the present invention is to provide a method for obtaining a full-length sequence of a template DNA by applying the above-mentioned tagged hairpin primer to amplification and labeling of a template DNA of unknown sequence or known sequence and then sequencing.
Compared with the prior art, the invention has the following beneficial effects:
the invention designs a labeled hairpin primer for amplifying and marking template DNA, which can be used for isothermal amplification of a low-concentration DNA template with unknown or known sequence, wherein the DNA template can be derived from environmental DNA, human and animal and plant tissue cells, blood, body fluid, viruses, bacteria, archaea and the like in microorganisms. For example, the amplification and labeling of the genomic DNA of a single virus can be accomplished by isothermal amplification, and after sequencing, the whole genomic sequence of the single virus can be obtained. Solves the problem that the sequence specific primer or the random primer in the prior art can not be used for marking the amplified product by using a DNA sequence tag while amplifying DNA.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a block diagram of a tagged hairpin primer designed in example 1 of the invention;
FIG. 2 is a graph showing the results of isothermal amplification and PCR verification of genomic DNA of White Spot Syndrome Virus (WSSV) (lane M: DNA Marker; lane 1: isothermal amplification of non-nucleic acid ultrapure water using a tag hairpin Primer; lane 2: isothermal amplification of non-nucleic acid ultrapure water using a WSSV specific Primer 1; lane 3: isothermal amplification of non-nucleic acid ultrapure water using a WSSV specific Primer 2; lane 4: isothermal amplification of non-nucleic acid ultrapure water using a WSSV specific Primer 3; lane 5: isothermal amplification of viral DNA having 10 viral particles cleaved and released per microliter using a tag hairpin Primer; lane 6: isothermal amplification of non-nucleic acid ultrapure water using a WSSV specific Primer 1; isothermal amplification of non-nucleic acid ultrapure water having 10 viral particles cleaved and released per microliter; lane 7: isothermal amplification of viral particles having 10 viral particles cleaved and released by WSSV specific Primer 2; and 8: isothermal amplification of viral particles having 10 viral particles cleaved and released by PCR Primer 3).
Detailed Description
In the following description, for purposes of explanation and not limitation, specific details are set forth such as the particular system architecture, techniques, etc., in order to provide a thorough understanding of the embodiments of the present invention. It will be apparent, however, to one skilled in the art that the present invention may be practiced in other embodiments that depart from these specific details.
Example 1
1. Experimental materials and methods
1. Experimental materials and reagents
Prawn White Spot Syndrome Virus (WSSV), tagged hairpin primers (100. Mu.M), annealing Buffer for DNA Oligos (5×), proteinase K, reaction buffer, non-nucleic acid ultrapure water, dNTP, phi29 polymerase, three pairs of WSSV genome-specific primers (Primer 1,5'-GAACTCCTGGCAAGGTATT-3' and 5'-GGTGTATCGTCAGCAAGAA-3'; primer2,5'-CCTTAGGCGAGTCATGTT-3' and 5'-TTGGTTGTGGTTGTGGAT-3'; primer3,5'-TGACTGCTGAGGTTGGAT-3' and 5'-GAAGGAGGAGGTGTTGGA-3').
2. Experimental method
2.1 design of tagged hairpin primers with hairpin structures
By utilizing the principle of base complementary pairing, DNA tag sequences capable of forming 2 or 4 hairpin structures are respectively designed, and the hairpin structures are connected with an artificially designed 6-base random primer (the sequence is 3' -NNNNNNNNN) to form a tag hairpin primer with the hairpin structures, as shown in figure 1.
2.2 tag hairpin primers to obtain hairpin structures
Carrying out high-temperature annealing treatment on the designed tag hairpin primer, wherein an annealing system comprises the following components: 40 μl of the tagged hairpin primer (100 μM), 10 μ l Annealing Buffer for DNA Oligos (5×). The required annealing procedure was 95℃for 5min; decreasing the temperature to 25 ℃ every 1min by 1 ℃; preserving heat at 4 ℃ to finally obtain the tag hairpin primer with the hairpin structure.
2.3 isothermal amplification, labeling and validation
2.3.1 isothermal amplification and labeling of viral genomic DNA of the White Spot Syndrome Virus (WSSV) with the obtained tagged hairpin primers:
the method comprises the following specific steps: purifying to obtain virus particles of White Spot Syndrome Virus (WSSV), and taking the virus particles as a DNA template, and infinitely diluting the WSSV virus until the virus particles contain 10 virus particles per microliter; cleaving the virus by proteinase K (56 ℃,1 h) to release DNA; 1. Mu.l of DNA template, 1. Mu. l reaction buffer (reaction buffer) and 4. Mu.l of non-nucleic acid ultrapure water were added to the PCR tube and reacted at 95℃for 3min; placing on ice for 10min; 2.5. Mu.l of the tagged hairpin primer (100. Mu.M), 1. Mu.l of NTP (100. Mu.M) and 0.5. Mu.l of Phi29 polymerase after the high temperature annealing treatment were added to the reaction mixture in the first step, and the temperature was 30℃for 3 to 6 hours; the polymerase was inactivated by reaction at 65℃for 10 min. Meanwhile, setting non-nucleic acid ultrapure water as negative control, and adopting proteinase K (56 ℃ C., 1 h) for treatment according to the same steps; 1. Mu.l of non-nucleic acid ultrapure water template, 1. Mu. l reaction buffer and 4. Mu.l of non-nucleic acid ultrapure water are added into a PCR tube and reacted for 3min at 95 ℃; placing on ice for 10min; 2.5. Mu.l of the tagged hairpin primer (100. Mu.M), 1. Mu.l of dNTP (100. Mu.M) and 0.5. Mu.l of Phi29 polymerase after the high temperature annealing treatment are added to the reaction mixture in the first step, and the temperature is 30 ℃ for 3 to 6 hours; the polymerase was inactivated by reaction at 65℃for 10 min.
2.3.2 PCR verification of isothermal amplification products
Three pairs of WSSV specific primers (Primer 1, primer2 and Primer 3) are designed according to different positions on the WSSV genome, PCR verification is carried out by taking the product after isothermal amplification as a template, and if PCR is carried out by using the three pairs of primers, PCR products can be obtained, thus indicating that the isothermal amplification products cover the WSSV full-length genome. The PCR system used was: 0.5. Mu.l of DNA template, 6. Mu.l of Taq enzyme, 4.5. Mu.l of non-nucleic acid ultrapure water, 0.5. Mu.l of WSSV specific primer front primer, 0.5. Mu.l of WSSV specific primer rear primer. The PCR procedure used was: 95 ℃ for 5min;35 cycles (95 ℃,10s;55 ℃,30s;72 ℃,40 s); 72 ℃ for 10min; preserving heat at 4 ℃.
As shown in FIG. 2, lanes 1 to 4 are negative controls using non-nucleic acid ultrapure water as a template, lane 1 is the result of isothermal amplification of non-nucleic acid ultrapure water using a labeled hairpin Primer, lanes 2 to 4 are the result of PCR amplification of isothermal amplification products of non-nucleic acid ultrapure water using three pairs of WSSV specific primers, respectively (Primer 1 is used in lane 2, primer2 is used in lane 3, and Primer3 is used in lane 4); lane 5 shows isothermal amplification of viral DNA released by cleavage of 10 virions per microliter using a tagged hairpin primer; lanes 6-8 are the results of PCR amplification using three pairs of WSSV specific primers for each microliter of isothermal amplification products containing 10 viral DNA released by viral particle cleavage (lane 6 using Primer1, lane 7 using Primer2, lane 8 using Primer 3).
As can be seen from the results of FIG. 2, when the isothermal amplification is performed by diluting the virus to 10 virus particles per liter using the tagged hairpin primer of the invention, isothermal amplification products of a low concentration of template can be obtained; PCR verification is carried out by adopting three pairs of WSSV specific primers respectively, corresponding fragment bands (lanes 5-8) can be obtained, and the isothermal amplification products are shown to cover the WSSV full-length genome; therefore, the WSSV genome can be amplified and labeled by isothermal amplification using the labeled hairpin primers designed by the invention. Comparing with the negative control (lanes 1-4), no DNA band was seen in the negative control lanes, indicating no isothermal amplification product, and PCR verification was performed on the equivalent Wen Kuozeng product by using three pairs of WSSV specific primers, respectively, and no obvious band was observed, indicating that the labeled hairpin primers designed by the invention cannot amplify and label templates of non-nucleic acid ultrapure water. Taken together, the experimental results show that the labeled hairpin primers designed by the invention can be used for DNA amplification and labeling of WSSV with low concentration.
The present invention is not limited to the above-described specific embodiments, and various modifications may be made by those skilled in the art without inventive effort from the above-described concepts, and are within the scope of the present invention.
Claims (7)
1. A tagged hairpin primer for DNA amplification and labeling, characterized in that the tagged hairpin primer comprises a DNA tag sequence with 2 or more hairpin structures at the 5' end and a 6-base random primer.
2. The tagged hairpin primer for DNA amplification and labeling of claim 1, wherein the hairpin structure in the tagged hairpin primer is directly linked to a 6-base random primer or is linked through a base.
3. A method for DNA amplification and labeling using the tagged hairpin primer of claim 1 or 2, comprising the steps of:
s1, extracting DNA from a sample to be detected, and taking the DNA as template DNA;
s2, carrying out isothermal amplification reaction on the template DNA by using the labeled hairpin primer and an isothermal amplification reaction system to obtain a labeled isothermal amplification product.
4. The method for amplifying and labeling DNA by using the labeled hairpin primer according to claim 3, wherein in the step S2, the composition of the isothermal amplification reaction system comprises: template DNA, phi29 polymerase, reaction buffer, dntps, tagged hairpin primers, and nucleotide-free ultrapure water.
5. The method for amplifying and labeling DNA by using the labeled hairpin primer according to claim 3, wherein the isothermal amplification reaction procedure is as follows: 30 ℃ for 3-6h;65 ℃ for 10min; preserving heat at 4 ℃.
6. The method for amplifying and labeling DNA by using the labeled hairpin primer according to claim 3, wherein the sample to be tested is derived from environmental DNA, human and animal tissue cells, blood, body fluid, or viruses, bacteria and archaebacteria in microorganisms.
7. The tagged hairpin primer of claim 1 which is applied to full-length sequence amplification and sequencing of template DNA of unknown sequence or known sequence, and the full-length sequence of the template DNA is obtained.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1836050A (en) * | 2003-07-07 | 2006-09-20 | 单细胞系统公司 | Hairpin-labeled probes and methods of use |
| CN101213311A (en) * | 2005-04-29 | 2008-07-02 | J·克雷格·文特尔研究院 | Amplification and cloning of single DNA molecules using rolling circle amplification |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1836050A (en) * | 2003-07-07 | 2006-09-20 | 单细胞系统公司 | Hairpin-labeled probes and methods of use |
| CN101213311A (en) * | 2005-04-29 | 2008-07-02 | J·克雷格·文特尔研究院 | Amplification and cloning of single DNA molecules using rolling circle amplification |
Non-Patent Citations (1)
| Title |
|---|
| 张越: "多重置换扩增在病理切片DNA分型中的应用", 《法医学杂志》, vol. 29, no. 1, pages 17 - 20 * |
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