CN110603328A - Quantitative PCR amplification primer pair and application thereof - Google Patents
Quantitative PCR amplification primer pair and application thereof Download PDFInfo
- Publication number
- CN110603328A CN110603328A CN201780090496.8A CN201780090496A CN110603328A CN 110603328 A CN110603328 A CN 110603328A CN 201780090496 A CN201780090496 A CN 201780090496A CN 110603328 A CN110603328 A CN 110603328A
- Authority
- CN
- China
- Prior art keywords
- primer
- pcr amplification
- quantitative pcr
- sequence
- amplification
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 230000003321 amplification Effects 0.000 title claims abstract description 183
- 238000003199 nucleic acid amplification method Methods 0.000 title claims abstract description 183
- 238000003753 real-time PCR Methods 0.000 title claims abstract description 105
- 230000000295 complement effect Effects 0.000 claims abstract description 22
- 238000010791 quenching Methods 0.000 claims abstract description 10
- 230000000171 quenching effect Effects 0.000 claims abstract description 10
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 5
- 238000000034 method Methods 0.000 claims description 95
- 238000006243 chemical reaction Methods 0.000 claims description 23
- 238000004445 quantitative analysis Methods 0.000 claims description 22
- 230000014509 gene expression Effects 0.000 claims description 16
- 238000000137 annealing Methods 0.000 claims description 13
- 238000004458 analytical method Methods 0.000 claims description 12
- 108090000623 proteins and genes Proteins 0.000 claims description 11
- 238000012986 modification Methods 0.000 claims description 10
- 238000012360 testing method Methods 0.000 claims description 10
- 230000004048 modification Effects 0.000 claims description 9
- 238000012197 amplification kit Methods 0.000 claims description 7
- 230000026731 phosphorylation Effects 0.000 claims description 4
- 238000006366 phosphorylation reaction Methods 0.000 claims description 4
- 125000000446 sulfanediyl group Chemical group *S* 0.000 claims description 4
- 239000002299 complementary DNA Substances 0.000 claims description 3
- 108091093037 Peptide nucleic acid Proteins 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- 108020004414 DNA Proteins 0.000 description 78
- 239000000523 sample Substances 0.000 description 56
- 238000011529 RT qPCR Methods 0.000 description 55
- 239000000047 product Substances 0.000 description 44
- 238000012163 sequencing technique Methods 0.000 description 24
- 238000012408 PCR amplification Methods 0.000 description 23
- 238000001514 detection method Methods 0.000 description 20
- 210000002700 urine Anatomy 0.000 description 19
- 108020004638 Circular DNA Proteins 0.000 description 13
- 239000000539 dimer Substances 0.000 description 12
- 238000001917 fluorescence detection Methods 0.000 description 12
- 239000012634 fragment Substances 0.000 description 11
- 230000027455 binding Effects 0.000 description 10
- 238000013461 design Methods 0.000 description 10
- 230000000694 effects Effects 0.000 description 9
- 210000003754 fetus Anatomy 0.000 description 9
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 108091093088 Amplicon Proteins 0.000 description 6
- 102000053602 DNA Human genes 0.000 description 5
- 210000002593 Y chromosome Anatomy 0.000 description 5
- 239000000975 dye Substances 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 102000003960 Ligases Human genes 0.000 description 4
- 108090000364 Ligases Proteins 0.000 description 4
- CGNLCCVKSWNSDG-UHFFFAOYSA-N SYBR Green I Chemical compound CN(C)CCCN(CCC)C1=CC(C=C2N(C3=CC=CC=C3S2)C)=C2C=CC=CC2=[N+]1C1=CC=CC=C1 CGNLCCVKSWNSDG-UHFFFAOYSA-N 0.000 description 4
- 230000000875 corresponding effect Effects 0.000 description 4
- 239000007850 fluorescent dye Substances 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000007363 ring formation reaction Methods 0.000 description 4
- 238000007400 DNA extraction Methods 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108060002716 Exonuclease Proteins 0.000 description 3
- 238000004925 denaturation Methods 0.000 description 3
- 230000036425 denaturation Effects 0.000 description 3
- 238000012938 design process Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 102000013165 exonuclease Human genes 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000007689 inspection Methods 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 230000002194 synthesizing effect Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 2
- 230000001605 fetal effect Effects 0.000 description 2
- 238000002844 melting Methods 0.000 description 2
- 230000008018 melting Effects 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 230000004568 DNA-binding Effects 0.000 description 1
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 1
- 108010010677 Phosphodiesterase I Proteins 0.000 description 1
- 239000013614 RNA sample Substances 0.000 description 1
- 238000004873 anchoring Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000013211 curve analysis Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000003205 genotyping method Methods 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000007837 multiplex assay Methods 0.000 description 1
- 239000002751 oligonucleotide probe Substances 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 238000005381 potential energy Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000011897 real-time detection Methods 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000020509 sex determination Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Plant Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
There is provided a quantitative PCR amplification primer pair comprising: a first primer and a second primer, wherein the first primer comprises a first specific sequence and a first random sequence, the first specific sequence is located at the 3 'end of the first primer, the first random sequence is located at the 5' end of the first primer, the second primer comprises a second specific sequence and a second random sequence, the second specific sequence is positioned at the 3 'end of the second primer, and the second random sequence is positioned at the 5' end of the second primer, and the first specific sequence and the second specific sequence are respectively an upstream primer and a downstream primer aiming at the target sequence, and the first random sequence and the second random sequence are reversely complementary, the 5 'end base of the first primer and the 5' end base of the second primer are both connected with a fluorescent group, and a quenching group is connected to the base with a specific length away from the 5 'terminal base of the first primer and the 5' terminal base of the second primer.
Description
PRIORITY INFORMATION
Is free of
The invention relates to the technical field of biology, in particular to quantitative PCR amplification, and more particularly relates to a quantitative PCR amplification primer pair and application thereof.
The real-time fluorescence quantitative PCR realizes the quantitative and qualitative analysis of the initial template by detecting the fluorescence signal of each cycle product in the PCR amplification reaction in real time. In the real-time fluorescent quantitative PCR reaction, a fluorescent chemical substance is introduced, and as the PCR reaction proceeds, the PCR reaction products are accumulated continuously, and the fluorescent signal intensity is increased in equal proportion. After each cycle, a fluorescence intensity signal is collected, so that the change of the product amount can be monitored through the change of the fluorescence intensity, and a fluorescence amplification curve graph is obtained.
The real-time fluorescence quantitative PCR technology is a leap of the DNA quantitative technology. By using the technology, the DNA and RNA samples can be quantitatively and qualitatively analyzed.
However, the current real-time fluorescent quantitative PCR technology still needs to be improved.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, an object of the present invention is to provide a real-time fluorescent quantitative PCR technique with high amplification efficiency and good specificity, and a corresponding PCR primer pair and probe composition.
First, it should be noted that the present invention has been completed based on the following findings and works of the inventors:
the most commonly used methods for qPCR are currently the SYBR-dependent dye method and the Taqman probe-dependent probe method; the former uses fluorescent dyes to indicate an increase in amplification, and the latter uses probes that specifically hybridize to the target sequence to indicate an increase in amplification product.
SYBR Green I is a double stranded DNA binding dye that binds in the minor groove. After binding to double-stranded DNA, its fluorescence is greatly enhanced. This property makes it ideal for detection of amplification products. In a PCR reaction system, excessive SYBR fluorescent dye is added, and after the SYBR fluorescent dye is specifically doped into a DNA double strand, a fluorescent signal is emitted, while SYBR dye molecules which are not doped into the strand do not emit any fluorescent signal, so that the increase of the fluorescent signal and the increase of a PCR product are completely synchronous. SYBR Green I has many advantages in real-time detection of nucleic acids, since it can bind to all double-stranded DNA and does not have to be specifically tailored for different templates, thus allowing for a very versatile design procedure and a relatively low cost. The fluorescent dye can indicate the property of the melting point of the double-stranded DNA, and the amplification product and the primer dimer can be identified through melting point curve analysis, so that non-specific amplification can be distinguished, and further, monochromatic multiplex determination can be realized. In addition, the sensitivity of SYBR Green I is high because one PCR product can be combined with multiple molecules of dye. However, since SYBR Green I binds to all double-stranded DNA, false positives due to primer dimers, single-stranded secondary structures, and false amplification products can affect the accuracy of quantitation.
The TaqMan probe is an oligonucleotide probe whose fluorescence is correlated with the amplification of a target sequence. It is designed to pair with the sequence between the upstream and downstream primers of the target sequence. The fluorophore is attached to the 5 'end of the probe and the quencher is at the 3' end. When the complete probe is paired with the target sequence, the fluorescence emitted by the fluorophore is quenched due to proximity to the quencher at the 3' end. However, when the extension reaction is carried out, the 5' exonuclease activity of the polymerase enzyme cleaves the probe, allowing the fluorophore to separate from the quencher. The released fluorophores accumulate as the number of amplification cycles increases. The fluorescence intensity is thus proportional to the amount of amplification product.
Although the Taqman probe method has strong specificity, a specific probe needs to be designed, the primer and the probe need to be optimized, and a better primer and probe combination is difficult to obtain in a certain complicated region. In multiplex assays, it is more difficult to optimize a good primer combination (primer pair and specific probe) and in some areas, although fluorescence detection is specific, it is still common to actually encounter the occurrence of non-specific PCR products, which will not generate fluorescence, but will affect the amplification efficiency of PCR primer pairs and the uniformity of different amplification areas, and will generate larger deviations in relative quantification. And there is often insufficient space to accommodate probes in some short template DNA molecules (e.g., urine free DNA, average molecular size 30-50 bp). The SYBR dye method does not require consideration of the template length, but has low specificity, and non-specific products and primer dimers interfere with fluorescence collection.
Therefore, there is a need to develop a new real-time fluorescent quantitative PCR method with good specificity and high quantitative accuracy.
Through a series of scientific researches and experimental explorations, the inventor unexpectedly discovers that: the primer pair (sometimes also referred to as "locked primer" (PP) herein) with a stable primer dimer structure with "5 ' reverse complementarity and 3 ' overhang" is formed by adding a complementary sequence to the 5 ' end of a pair of conventional primers, so that the specificity of PCR amplification can be effectively improved, and the amplification efficiency can be increased.
Furthermore, the inventors have found that, in the case of PCR amplification by designing a fluorescent group and a quencher group on the 5 'end of a lock primer based on the above-mentioned primer pair having a stable primer dimer structure in which the 5' end is reverse complementary and the 3 'end protrudes (i.e., "lock primer"), the primer is extended to form a loop and then the 5' end of the primer is cleaved to release the fluorescent group to a solution to generate fluorescence, and the fluorescence is detected to achieve quantitative or qualitative purposes. The method does not need to design a probe, so that the length of an amplicon which can be designed is shorter than that of the probe method; compared with a probe method, the method can generate two fluorescent molecules after synthesizing one template, so that the fluorescence intensity is greatly increased; compared with the existing qPCR technology, the method has the characteristics of strong specificity, high amplification efficiency, good uniformity and the like, has very good uniformity in qPCR multiple amplification, and has certain advantages for multi-site qPCR detection.
Thus, in a first aspect of the invention, the invention provides a quantitative PCR amplification primer pair. According to an embodiment of the present invention, the quantitative PCR amplification primer pair includes: a first primer and a second primer, wherein the first primer comprises a first specific sequence and a first random sequence, the first specific sequence is positioned at the 3 'end of the first primer, the first random sequence is positioned at the 5' end of the first primer, the second primer comprises a second specific sequence and a second random sequence, the second specific sequence is positioned at the 3 'end of the second primer, the second random sequence is positioned at the 5' end of the second primer, the first specific sequence and the second specific sequence are respectively an upstream primer and a downstream primer aiming at a target sequence, the first random sequence and the second random sequence are in reverse complementarity, the 5 'terminal base of the first primer and the 5' terminal base of the second primer are connected with a fluorescent group, and the base with a specific length from the 5 'terminal base of the first primer and the 5' terminal base of the second primer are connected with a quenching group . The inventor surprisingly finds that the qPCR performed by using the quantitative PCR amplification primer pair of the invention has stronger specificity, higher amplification efficiency, higher fluorescence intensity of products and low background noise compared with the conventional qPCR, and the result is more accurate and reliable when the quantitative PCR amplification primer pair is used for quantitative analysis of DNA or RNA and gene expression difference analysis. The quantitative PCR amplification primer pair has good uniformity when used for qPCR multiple amplification and has certain advantages for multi-site qPCR detection.
In a second aspect of the invention, the invention provides a quantitative PCR amplification kit. According to an embodiment of the invention, the kit comprises the pair of quantitative PCR amplification primers as described above. According to the embodiment of the invention, compared with the conventional qPCR, when the quantitative PCR amplification primer pair in the kit is used for qPCR, the specificity is stronger, the amplification efficiency is higher, the fluorescence intensity of the product is higher, the background noise is low, and when the quantitative PCR amplification primer pair is used for quantitative analysis of DNA or RNA and gene expression difference analysis, the result is more accurate and reliable. The quantitative PCR amplification primer pair has good uniformity when used for qPCR multiple amplification and has certain advantages for multi-site qPCR detection.
In a third aspect of the invention, the invention provides a method of quantitative PCR amplification. According to an embodiment of the present invention, the method performs the quantitative PCR amplification using the aforementioned quantitative PCR amplification primer pair or quantitative PCR amplification kit. Thus, quantitative PCR amplification of the template can be efficiently achieved by this method. Compared with the existing qPCR method, the quantitative PCR amplification method can increase the specificity of amplification, effectively reduce the generation of non-specific products, improve the amplification efficiency, increase the fluorescence intensity of the products and reduce background noise, thereby obviously increasing the accuracy of fluorescence detection and improving the accuracy of quantitative results.
In a fourth aspect of the invention, the invention provides a method for quantitative analysis of a test DNA sample. According to an embodiment of the invention, the method comprises: according to the quantitative PCR amplification method, the DNA sample to be detected is subjected to fluorescent quantitative PCR amplification, and quantitative analysis is realized based on the collected fluorescent signal. Therefore, the PCR amplification has good specificity, high amplification efficiency, high fluorescence detection accuracy and accurate and reliable quantitative analysis result.
In a fifth aspect of the present invention, the present invention provides a method for performing gene expression differential analysis of a specific gene on a plurality of DNA samples to be tested. According to an embodiment of the present invention, the plurality of test DNA samples each comprise a cDNA sequence of the specific gene, the method comprising: according to the quantitative PCR amplification method, the multiple DNA samples to be detected are respectively subjected to fluorescent quantitative PCR amplification, and quantitative analysis is realized based on the collected fluorescent signals; and comparing the quantitative analysis results of the plurality of DNA samples to be detected so as to determine the gene expression difference of the specific genes of the plurality of DNA samples to be detected. Therefore, the result of the gene expression difference analysis is accurate and reliable.
According to the embodiment of the invention, the quantitative PCR amplification primer pair and the application thereof have at least one of the following advantages:
1. the design strategy of the quantitative PCR amplification primer pair simplifies the primer design process and optimizes the experimental steps, the primer pair consists of a specific sequence at the 3 'end and a random sequence (complementary sequence) at the 5' end, and forward and reverse primers form a stable dimer structure through the complementary sequence without meeting strict conditions of conventional primers, thereby greatly simplifying the design process. The PCR primer pair can achieve a good amplification effect without special optimization at the 5' end, and the design time of the primer is short. In the conventional primer design process, the complementarity of the 5 'end and the 5' end of the primer, the formation of a palindrome structure by the primer itself, and the like need to be avoided, so as to ensure that a dimer structure cannot be formed between the primers and self-extension can occur for PCR. However, the design of the PCR primer pair of the present invention does not need to take these problems into consideration because the structure of the locking primer itself is such that the 5 'end is complementary to the stable dimer and the 3' end of the dimer can be extended by being complementary to the specific sequence normally, whereas the 3 'end of the conventional primer does not have enough sequence to bind to the specific complementary sequence if the 5' end forms the dimer structure. Moreover, the potential energy of the 5 'end complementary sequence formed between 2 primers of the locking primer is far greater than that of the self palindrome, so that the 5' end dimer structure can be preferentially formed even if the 3 'end and the 5' end have complementary sequences.
2. The specificity of PCR amplification can be increased by utilizing the quantitative PCR amplification primer pair to carry out PCR amplification, and the generation of non-specific products is effectively reduced; it starts from the second cycle of PCR, the base (random sequence) at the 5 'end of the primer and the base at the 5' end of the newly generated template are reversely and complementarily combined, the specific sequence at the 3 'end of the primer and the base at the 3' end of the newly generated template are reversely and complementarily combined, that is, the number of the primer and the template combined recognition sites is 2 (as shown in figure 2), thereby the combining ability of the primer and the template and the specificity of amplification are obviously increased. The combination rate of the primer and the template is improved, and the amplification efficiency is effectively improved.
3. The quantitative PCR amplification primer pair provided by the invention is used for PCR amplification, and the GC bias of different templates in the amplification of a sequencing library (especially a second-generation sequencing library) can be effectively reduced, because the PCR amplification can be effectively amplified only when the primers are combined after the template is denatured. GC bias arises because in some high GC regions, the template renatures very rapidly during PCR, and the template renatures without binding to the primers, rendering these regions ineffective for amplification. The locking primer and the template of the invention have 2 binding sites, so that the binding capacity with the template can be greatly improved, and the template with high GC content can be effectively bound, thereby reducing GC bias.
4. The quantitative PCR amplification method of the invention does not need to design a probe, directly utilizes the quantitative PCR amplification primer pair of the invention to carry out PCR amplification, removes the external cutting of the self fluorescent group at the 5' end after the primer is extended into a ring, generates fluorescence when the fluorescent group is dissociated into a solution, and can achieve the purpose of quantitative or qualitative detection through the detection of the fluorescence; the method does not need to design a probe, so that the length of an amplicon which can be designed is shorter than that of the probe method; in addition, a probe method generates one fluorescent molecule when synthesizing one template, and qPCR is carried out by using the quantitative PCR amplification primer pair of the invention, and two fluorescent molecules are generated after synthesizing one template, so that the invention greatly increases the fluorescence intensity of the product and reduces the background noise compared with the probe method, thereby obviously increasing the accuracy of fluorescence detection; unlike qPCR based on probe, the qPCR of the present invention needs no occupying too much template space and has great advantages for short segment template, i.e., the method of the present invention can also perform circular amplification for short segment template and can also achieve good quantitative or shape-fixed detection effect for short segment DNA.
5. The product obtained by the quantitative PCR amplification method is a nicked annuloid (namely, the 5 'end and the 3' end of the annuloid are not connected), and for experiments that some products need to be cyclized, only ligase is added to realize cyclization without complex denaturation and quenching processes, so that the experimental process can be effectively simplified.
6. The quantitative PCR amplification method of the present invention relies on a specially designed quantitative PCR amplification primer pair. Compared with the conventional qPCR, when the composition is used for qPCR, the specificity is stronger, the amplification efficiency is higher, the fluorescence intensity of a product is higher, the background noise is lower, and when the composition is used for quantitative analysis of DNA or RNA and gene expression difference analysis, the result is more accurate and reliable; the quantitative PCR amplification primer pair has good uniformity when used for qPCR multiple amplification and has certain advantages for multi-site qPCR detection.
7. The quantitative PCR amplification primer pair, the quantitative PCR amplification kit and the quantitative PCR amplification method can be widely applied to the fields of quantitative analysis of DNA or RNA, gene expression difference analysis, genotyping, pathogen detection and the like.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 shows a schematic diagram of the structure of a PCR primer pair (i.e., a locking primer) according to the present invention, according to one embodiment of the present invention;
FIG. 2 is a schematic diagram showing the binding of primers to a newly formed strand in the circular amplification of the present invention, according to an embodiment of the present invention;
FIG. 3 shows a schematic flow chart of quantitative PCR amplification using the quantitative PCR amplification primer pair of the present invention according to an embodiment of the present invention;
FIG. 4 shows the qPCR fluorescent signal detection results of the conventional SRY primer and the lock primer on the DNA sample to be tested in example 1;
FIG. 5 shows the results of the qPCR fluorescent signal detection of each sample in example 1.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.
It should be noted that the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Further, in the description of the present invention, "a plurality" means two or more unless otherwise specified.
Quantitative PCR amplification primer pair
In a first aspect of the invention, the invention provides a quantitative PCR amplification primer pair. According to an embodiment of the present invention, the quantitative PCR amplification primer pair includes: a first primer and a second primer, wherein the first primer comprises a first specific sequence and a first random sequence, the first specific sequence is positioned at the 3 'end of the first primer, the first random sequence is positioned at the 5' end of the first primer, the second primer comprises a second specific sequence and a second random sequence, the second specific sequence is positioned at the 3 'end of the second primer, the second random sequence is positioned at the 5' end of the second primer, the first specific sequence and the second specific sequence are respectively an upstream primer and a downstream primer aiming at a target sequence, the first random sequence and the second random sequence are in reverse complementarity, the 5 'terminal base of the first primer and the 5' terminal base of the second primer are connected with a fluorescent group, and the base with a specific length from the 5 'terminal base of the first primer and the 5' terminal base of the second primer are connected with a quenching group . The inventor surprisingly finds that by utilizing the quantitative PCR amplification primer pair, a probe is not required to be designed, the primer pair is directly utilized for PCR amplification, the 5' end of the primer pair is removed after the primer pair is extended into a ring, the fluorescent group is dissociated into a solution to generate fluorescence, and the aim of quantitative or qualitative detection can be achieved through the detection of the fluorescence; in addition, a probe method generates one fluorescent molecule every time one template is synthesized, and qPCR is performed by using the quantitative PCR amplification primer pair of the invention, and two fluorescent molecules are generated every time one template is synthesized, so that the invention greatly increases the fluorescent intensity compared with the probe method, thereby obviously increasing the accuracy of fluorescent detection. In addition, the quantitative PCR amplification primer pair can effectively reduce GC bias in the PCR amplification process and improve the amplification specificity. Specifically, a certain GC bias is brought in the process of enrichment of a second-generation sequencing library PCR by using a conventional primer, and when qPCR is carried out by using the quantitative PCR amplification primer pair with the locked primer structure, 2 primer-combined sites are arranged on a newly generated template in the second-step amplification of the PCR, so that the combining capacity of the primer and the template is greatly enhanced, the condition that the amplification efficiency is low due to high GC content and quick renaturation of the template and incapability of combining the primer is effectively reduced, namely, the GC bias in the PCR enrichment process can be effectively reduced, the problem of amplification bias among different templates can be effectively reduced by the same principle, and the problem of one-tube amplification of multiple primers is solved. Furthermore, when the quantitative PCR amplification primer pair is used for qPCR, compared with the conventional qPCR, the specificity is stronger, the amplification efficiency is higher, and when the quantitative PCR amplification primer pair is used for quantitative analysis of DNA or RNA and gene expression difference analysis, the result is more accurate and reliable. The quantitative PCR amplification primer pair has good uniformity when used for qPCR multiple amplification and has certain advantages for multi-site qPCR detection.
It should be noted that the "first random sequence" and the "second random sequence" in the present invention may be random sequences or fixed sequences, as long as the two sequences are complementary in reverse.
According to an embodiment of the invention, the TM value of the first and second specific sequences is 55-65 degrees celsius and the TM value of the first and second primers is 65-75 degrees celsius. Thus, the PCR can be performed first with a first round of linear amplification at a low annealing temperature (55-65 degrees Celsius), and then with a second round of cyclic amplification at a higher annealing temperature (65-72 degrees Celsius) in subsequent cycles. Because the round annealing temperature is high during the circular amplification process, the specific sequence can not be singly combined to the specific site (the TM value of the specific sequence is only 55-65 ℃), and the PCR can be effectively amplified only when the 5 ' end of the locking primer is combined to the 5 ' end of the template and the 3 ' end of the locking primer is combined to the specific site of the template, namely the round amplification is actually circular amplification of double binding sites.
The quantitative PCR amplification primer pair is suitable for carrying out PCR amplification and library construction on DNA samples to be detected in any forms. It should be noted that the "DNA sample to be tested" in the present invention is somewhat different from the conventional understanding that does not include the treated DNA. However, in the present invention, the "DNA sample to be tested" may include processed DNA and unprocessed DNA (in constructing a sequencing library, generally, a sample is subjected to breaking and sequencing linker treatment on genomic DNA to obtain DNA fragments carrying corresponding platform sequencing linkers, and after subsequent amplification steps, the obtained product may be used for sequencing, where the DNA fragments carrying corresponding platform sequencing linkers are "processed DNA fragments", and correspondingly, the DNA fragments are "unprocessed DNA" after the above-mentioned treatment). If the DNA is not processed, the PCR primer pair is used for amplification, namely the specific target segment is amplified; if the target fragment is a DNA fragment for the treated DNA, the target fragment to be amplified may be a DNA fragment of the entire genome.
According to some embodiments of the present invention, when the DNA sample to be tested is a processed DNA fragment carrying a universal sequence (e.g. a sequencing adaptor) (where "universal sequence" is a sequence for pairing with a specific sequence in a primer, including a sequencing platform adaptor sequence, i.e. a sequencing adaptor), the first specific sequence and the second specific sequence must be capable of specifically recognizing a target sequence carrying a universal sequence, in other words, the target sequence at this moment is actually "universal sequence + target region sequence"; when the DNA sample to be tested (i.e. the PCR reaction template) is a DNA fragment not carrying a universal sequence, the first specific sequence and the second specific sequence can specifically recognize the target sequence accordingly. In addition, if a sequencing library is required to be constructed, a sequencing linker sequence (i.e., a universal sequence) can be set in the random sequence of the first primer and the second primer or between the specific sequence and the random sequence, so that a sequencing linker is connected to a PCR amplification product, and the sequencing linker can be effectively used in a sequencing platform.
According to further embodiments of the present invention, at least one of the first primer and the second primer further comprises a tag sequence, whereby multiple samples can be simultaneously PCR amplified and the samples can be distinguished based on the tag sequence. The position of the tag sequence in the first primer and the second primer is not particularly limited, as long as the tag sequence can perform the function of distinguishing each sample and does not affect the progress of PCR amplification. According to some specific examples of the invention, the tag sequence may be located between the specific sequence and the random sequence, whereby one tag sequence may be located between the first specific sequence and the first random sequence of the first primer and/or one tag sequence may be located between the second specific sequence and the second random sequence of the second primer. According to another embodiment of the present invention, the tag sequence may also be configured to be included in a random sequence, i.e. to be part of a random sequence. This also allows the sample to be distinguished from another sample, and does not affect the progress of PCR amplification.
According to an embodiment of the present invention, the length of the first random sequence and the second random sequence is 16-30bp, and the length of the first specific sequence and the second specific sequence is 16-30 bp.
According to an embodiment of the present invention, 2 nd to 5 th bases of 5 'ends of the first primer and the second primer are thio-modified, and 1 st to 5 th bases of 3' ends of the first primer and the second primer are thio-modified. This can effectively prevent the enzyme from being exogenously cleaved.
According to some embodiments of the present invention, the kind of thio modification is not particularly limited as long as the first primer and the second primer can be prevented from being exo-enzymatically (e.g., degraded by an enzyme having 5 '-3' or 3 '-5' exonuclease activity). According to some specific examples of the present invention, the thio modification is any one selected from the group consisting of a phosphorothioate-type modification, a methylsulfate-type modification and a peptide nucleic acid modification.
According to an embodiment of the present invention, the 5' end of at least one of the first primer and the second primer is modified by phosphorylation. Thus, the nicked circularity-like object obtained by two rounds of amplification (i.e., the 5 'end and the 3' end of the circularity-like object are not ligated) is ligated by adding a ligase to perform ligation reaction to form complete circular DNA. That is, circular DNA libraries can be prepared based on the products of qPCR.
According to some embodiments of the invention, the quencher group is attached to a base which is 10-30bp from the 5 'terminal base of the first primer and the 5' terminal base of the second primer.
As can be seen from the schematic structural diagram of the quantitative PCR amplification primer set of the present invention in fig. 1, the design strategy of the quantitative PCR amplification primer set of the present invention is: adding a complementary sequence to the 5 ' end of a pair of conventional primers (including a forward primer and a reverse primer) aiming at a target region, wherein the complementary sequence can be a random sequence or a fixed sequence, so that a pair of primers for PCR is designed to be reverse complementary at the 5 ' end, a ' locked primer ' (PP) protrudes from the 3 ' end, and a stable primer dimer structure is formed between the primer pairs of the locked primers; introducing a fluorescent group into the first base (namely the base at the 5 'end) of the primer at the 5' end, and introducing a quenching group into the base which is 10-30bp away from the base; in addition, 2 to 5 bases of the 5 'end of the primer may be thio-modified and 1 to 5 bases of the 3' end of the primer may be thio-modified to prevent degradation by 5 '-3' end exonuclease activity. The length of each strand of the whole primer pair is 32-60bp, the TM value is higher (the TM value of the first primer and the second primer is generally 65-75 ℃), wherein the length of the complementary sequence (namely the first random sequence and the second random sequence) at the 5' end of the locking primer is 16-30bp, and the sequence can be random or fixed; the length of the 3' end (i.e. the first specific sequence and the second specific sequence) is 16-30bp, which is complementary to the target sequence of the template, and the TM value is low (generally 55-65 ℃).
In addition, in order to solve the problem of the application of the quantitative PCR amplification primer set of the present invention, the quantitative PCR amplification primer set of the present invention requires 2 different amplification parts (i.e., two rounds of amplification) to perform PCR amplification (see fig. 3): in the first amplification part, the annealing temperature is 55-65 ℃, and the cycle number is 1; in the second amplification part, the annealing temperature is 65-72 ℃ and the number of cycles is 40-50. In the first amplification part, the PCR primer pair can only be combined with the template through the specific sequence at the 3' end, so the annealing temperature of the cycle is low; in the second amplification part, the PCR primer pair is firstly combined with the newly generated template (i.e. the product of the first round of amplification) through the 5 'end complementary sequences (i.e. the first random sequence and the second random sequence) and then combined with the newly generated template through the 3' end specific sequences (i.e. the first specific sequence and the second specific sequence), namely the primer and the template are combined with 2 recognition sites, and the combination of 2 anchoring sites greatly improves the annealing temperature of the primer, so the annealing temperature is higher.
In addition, it should be noted that in the second amplification part, the 5 'end and the 3' end of the primer can be effectively amplified circularly only when being combined with the newly generated template at the same time, so that the specificity of the PCR amplification is greatly improved by 2 recognition sites, and the binding capacity of the primer and the template is greatly improved by 2 binding sites, thereby improving the amplification efficiency of the PCR. Therefore, compared with the traditional PCR primer, the quantitative PCR amplification primer pair disclosed by the invention is used for PCR amplification, so that the specificity of PCR amplification can be obviously increased, the generation of non-specific products is effectively reduced, and the GC bias in the amplification process is reduced. Therefore, the primer is applied to sequencing, particularly to a second-generation sequencing library, and the GC bias in the whole genome range in library enrichment amplification can be effectively reduced.
Regarding the fluorescence detection mode when qPCR is performed using the quantitative PCR amplification primer set of the present invention: in the first amplification part, the primer at the 3 ' end is combined with the target complementary region, only the 3 ' end is combined with the target region, the annealing temperature is low, the polymerase extends, and the linear amplification is realized, only the template combination and extension are involved, the fluorescent group at the 5 ' end of the primer is not cut off, and therefore, fluorescence is not generated; in the second amplification part, the primer and the template have two binding sites, the 5 ' end of the primer is complementary to the 5 ' end of the newly generated strand (i.e., the product of the first round of amplification), the 3 ' end specific primer is bound to the target complementary region of the newly generated strand to form a loop, then the 3 ' end is extended, when the 3 ' end of the primer is extended to the 5 ' end, the 5 ' -3 ' end exo-activity of the polymerase will excise the base with the fluorescent group at the 5 ' end, the fluorescent group will generate fluorescence after dissociating into the solution, and the quantification can be realized by detecting the fluorescent molecule. During the qPCR, the two-step amplification of the two strands of the template occurs, so that two fluorescent molecules are generated every time one template is synthesized, thereby greatly increasing the intensity of fluorescence and reducing background noise.
Furthermore, according to the embodiments of the present invention, PCR amplification using the PCR primer pair of the present invention can be applied to product circularization, and the obtained products can be directly ligated to obtain circular DNA. Specifically, no additional steps such as denaturation and quenching are needed, and cyclization can be performed only by adding the product into a ligation reaction system, so that the cyclization process can be greatly simplified, and the preparation process of the circular DNA library is further simplified.
According to the embodiment of the invention, the sequence length of the specific probe is 18-30bp, and the TM value is 70-80 ℃.
Applications of
In a second aspect of the invention, the invention also provides a quantitative PCR amplification kit. According to an embodiment of the invention, the kit comprises the pair of quantitative PCR amplification primers as described above. According to the embodiment of the invention, compared with the conventional qPCR, when the quantitative PCR amplification primer pair in the kit is used for qPCR, the specificity is stronger, the amplification efficiency is higher, the fluorescence intensity of the product is higher, the background noise is low, and when the quantitative PCR amplification primer pair is used for quantitative analysis of DNA or RNA and gene expression difference analysis, the result is more accurate and reliable. The quantitative PCR amplification primer pair has good uniformity when used for qPCR multiple amplification and has certain advantages for multi-site qPCR detection.
Furthermore, the invention provides an application of the quantitative PCR amplification primer pair and a kit containing the same.
In a third aspect of the invention, the invention provides a method of quantitative PCR amplification. According to an embodiment of the present invention, the method performs the quantitative PCR amplification using the aforementioned quantitative PCR amplification primer pair or quantitative PCR amplification kit. Thus, quantitative PCR amplification of the template can be efficiently achieved by this method. Compared with the existing qPCR method, the quantitative PCR amplification method can increase the specificity of amplification, effectively reduce the generation of non-specific products, improve the amplification efficiency, increase the fluorescence intensity of the products and reduce background noise, thereby obviously increasing the accuracy of fluorescence detection and improving the accuracy of quantitative results.
According to an embodiment of the invention, the method comprises two rounds of amplification as follows: carrying out first round linear amplification on the quantitative PCR amplification primer pair and the template at an annealing temperature of 55-65 ℃; and performing a second round of circular amplification on the product of the first round of linear amplification at an annealing temperature of 65-72 ℃. Thus, starting from the second cycle of PCR (i.e., the second round of amplification), the 5 'base of the first primer and the second primer is reverse-complementarily combined with the 5' base of the newly generated template, and the 3 'specific base of the first primer and the second primer is reverse-complementarily combined with the 3' base of the newly generated template, i.e., the number of the primer and template binding recognition sites is 2 (as shown in FIG. 2), so that the specificity of PCR amplification can be increased, and the generation of non-specific products can be effectively reduced.
According to an embodiment of the present invention, the amplification reaction procedure of the method is as follows:
therefore, the PCR amplification process has low GC bias, high amplification specificity, good amplification effect and good fluorescence detection effect.
In a fourth aspect of the invention, the invention provides a method for quantitative analysis of a test DNA sample. According to an embodiment of the invention, the method comprises: according to the quantitative PCR amplification method, the DNA sample to be detected is subjected to fluorescent quantitative PCR amplification, and quantitative analysis is realized based on the collected fluorescent signal. Therefore, the PCR amplification has good specificity, high amplification efficiency, high fluorescence detection accuracy and accurate and reliable quantitative analysis result.
In a fifth aspect of the present invention, the present invention provides a method for performing gene expression differential analysis of a specific gene on a plurality of DNA samples to be tested. According to an embodiment of the present invention, the plurality of test DNA samples each comprise a cDNA sequence of the specific gene, the method comprising: according to the quantitative PCR amplification method, the multiple DNA samples to be detected are respectively subjected to fluorescent quantitative PCR amplification, and quantitative analysis is realized based on the collected fluorescent signals; and comparing the quantitative analysis results of the plurality of DNA samples to be detected so as to determine the gene expression difference of the specific genes of the plurality of DNA samples to be detected. Therefore, the result of the gene expression difference analysis is accurate and reliable.
In addition, the product obtained by the quantitative PCR amplification method is a similar ring with a notch (namely the 5 'end and the 3' end of the similar ring are not connected), and for experiments that some products need to be cyclized, only ligase is added to realize cyclization without complex denaturation and quenching processes, so that the experimental process can be effectively simplified.
Furthermore, in another aspect of the present invention, the present invention also provides a method for preparing a circular DNA library. According to an embodiment of the invention, the method comprises the steps of:
(1) subjecting a DNA sample to be tested to quantitative PCR amplification according to the aforementioned quantitative PCR amplification method, so as to obtain an amplification product comprising a loop-like material, wherein the 5 ' end and the 3 ' end of the loop-like material are not ligated, the 5 ' end of at least one of the first primer and the second primer is phosphorylation-modified, the 2 nd to 5 th bases of the 5 ' ends of the first primer and the second primer are thio-modified, and the 1 st to 5 th bases of the 3 ' ends of the first primer and the second primer are thio-modified; and
(2) subjecting the amplification products to a ligation reaction using a ligase to ligate the 5 'and 3' ends of the circularity-like objects to form circular DNAs, all of which constitute the circular DNA library,
wherein, when the 5 'end of one of the first primer and the second primer is modified by phosphorylation, the circular DNA library is a single-stranded circular DNA library, and when the 5' ends of the first primer and the second primer are both modified by phosphorylation, the circular DNA library is a double-stranded circular DNA library.
According to the embodiment of the invention, the method can be used for effectively preparing the single-stranded or double-stranded circular DNA library, and the obtained single-stranded or double-stranded circular DNA library has good library quality and good effect when being used for DNA storage or library sequencing.
According to an embodiment of the present invention, further comprising: (3) linear DNA was removed. Thus, the obtained library has good quality.
According to some specific examples of the invention, linear DNA is removed using a linear digestion reaction.
According to an embodiment of the present invention, in step (1), universal sequences are added to both ends of the DNA sample to be tested. As previously mentioned, the expression "universal sequence" as used herein refers to a sequence that is used to pair with a specific sequence in a primer, including sequencing platform linker sequences, i.e., sequencing linkers. Therefore, when universal sequences such as sequencing adapters are added to the two ends of the DNA sample to be tested, the obtained library can be directly used for the corresponding sequencing platform to perform on-machine sequencing.
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are carried out according to techniques or conditions described in literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruke et al, Huang Petang et al) or according to product instructions. The reagents or apparatus used are not indicated by the manufacturer, but are conventional products available commercially, for example from Illumina.
Example 1: method for detecting free DNA of fetus in urine by using locking primer method and Taqman probe method
1. Extracting free DNA of urine:
50ml of morning urine of a pregnant woman who was born as a fetus for 16 weeks was collected, and free DNA was extracted from the urine of the pregnant woman using Qiagen free DNA extraction Kit (QIAamp Circulating Nucleic Acid Kit, Cat number/ID: 55114) according to the protocol. And the obtained free DNA is subjected to qbait quantitative quality inspection. The quality test results are as follows:
table 1 free DNA concentration:
qPCR reaction:
the obtained free DNA is respectively subjected to qPCR by using a conventional Taqman probe method and a locking primer method (qPCR is carried out by using the quantitative PCR amplification primer pair of the invention), and the specific gene SRY on the Y chromosome is detected. Where the test was run in triplicate with water as the control.
Wherein, the two methods are performed by using Premix Ex Taq from takaraTMThe (cat No. RR390A) kit carries out qPCR reaction, and the reaction systems of the two methods are respectively as follows:
taqman probe method:
the locked primer method:
wherein the sequences of the primers SRY F and SRY R, the Probe SRY-Probe, the primer PP-SRY F and the primer PP-SRY R are shown in the following table 5.
Both methods were run on a qPCR reaction on a Stepone from ABI with the same reaction conditions as follows:
the flow of qPCR amplification using the PP primers of the invention is shown in FIG. 3.
The results of the amplified fluorescence detection signals are shown in Table 2 and FIG. 4.
Table 2 CT values detected by two methods:
as shown in FIG. 4, the urine fetus specific DNA fragment is smaller, and the qPCR amplification product is smaller under the condition of the same DNA amount, the specific fetal urine DNA molecules which can be utilized by the qPCR are more, so that the CT value detected by the locking primer (amplification product 43bp) is lower than the CT value detected by the Taqman probe method (amplification product 79 bp).
And (4) conclusion: because urine DNA is highly fragmented, there is a requirement for amplicons, which are shorter and more available DNA molecules under the same template. The Taqman probe method requires a probe design, that is, a certain space is reserved for the probe to bind to the length of the amplicon, compared to the method of locking the primer, and the locking primer does not require a probe design, thus having advantages in terms of the length of the amplicon. The short amplicon is important for fetal sex determination by early maternal urine DNA.
Example 2: detection of fetus specific Y chromosome in free DNA of urine fetus of early pregnant woman by using multiple pairs of primers
1. Urine DNA extraction
10ml of morning urine of pregnant women in 10 weeks is collected, wherein five cases of the morning urine of men and women are extracted by a Qiagen urine free DNA extraction Kit (QIAamp Circulating Nucleic Acid Kit, Cat number/ID: 55114) according to an operation instruction, the obtained DNA is subjected to qbuit quantitative quality inspection, and the subsequent qPCR reaction is carried out after the quality inspection is qualified. The quality test results are shown in Table 3.
Table 3 urine free DNA concentration:
qPCR reaction
Two pairs of locking primers are designed on specific genes SRY and DSY14 on a Y chromosome, and a pair of locking primers are designed on a GADPH gene on an autosome as an experiment and quantitative control, wherein fluorescent groups and quenching groups marked by the primers on the Y chromosome are FAM and BHQ1 respectively, and fluorescent groups and quenching groups marked by probes on the GADPH gene are HEX and BHQ1 respectively.
The 3 pairs of primers are used for carrying out multiplex qPCR amplification on the obtained DNA sample according to a locking primer method (qPCR is carried out by adopting the quantitative PCR amplification primer pair of the invention) so as to identify the urine free DNA fetus specific Y chromosome. With water as the control in this test.
The method comprises the following specific steps:
using Premix Ex Taq from TakaraTMThe kit (cat No. RR390A) was used for qPCR reactions in the following reaction systems:
the locked primer method:
remarking: the forward primer mix (10. mu.M) refers to: mixture of PP-SRY F, PP-DSY14F and PP-GADPH F, each primer concentration being 10. mu.M;
the reverse primer mix (10 μ M) refers to: PP-SRY R, PP-DSY14R and PP-GADPH R mixtures, each primer concentration was 10. mu.M.
The qPCR reaction was performed on a Stepone from ABI with the following reaction strip:
the flow of qPCR amplification using the PP primers of the invention is shown in FIG. 3.
The results of the detection of the amplified fluorescence detection signal are shown in Table 4 and FIG. 5.
Table 4 fluorescence detection CT values:
FIG. 5 shows fluorescence detection signals for simultaneous detection of FAM and HEX fluorescence.
And (4) conclusion: HEX fluorescence can be detected in 10 samples, and the difference of CT values is small, which indicates that cfDNA is extracted successfully and the difference of the total cfDNA amount is small; no fluorescence was detected in the water; FAM fluorescence can be detected in all five urine samples of male fetus, and FAM fluorescence is not detected in all five urine samples of female fetus; the method can detect the gender of fetus by using free DNA in urine of pregnant women of 8 weeks.
Table 5 qPCR primer sequences:
remarking: f: a forward primer; r: reverse primer, boxed with the complementary sequence.
The quantitative PCR amplification primer pair can be effectively used for quantitative PCR amplification of a DNA sample to be detected, has stronger specificity, higher amplification efficiency, higher fluorescence intensity of a product and low background noise compared with the conventional qPCR, and has more accurate and reliable results when being used for quantitative analysis of DNA or RNA and gene expression difference analysis. The quantitative PCR amplification primer pair has good uniformity when used for qPCR multiple amplification and has certain advantages for multi-site qPCR detection.
Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Various modifications and substitutions of those details may be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an illustrative embodiment," "an example," "a specific example," or "some examples" or the like mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
Claims (13)
- A quantitative PCR amplification primer pair, comprising:a first primer and a second primer, wherein the first primer and the second primer are different,wherein the content of the first and second substances,the first primer comprises a first specific sequence and a first random sequence, the first specific sequence is positioned at the 3 'end of the first primer, and the first random sequence is positioned at the 5' end of the first primer,the second primer comprises a second specific sequence and a second random sequence, the second specific sequence is positioned at the 3 'end of the second primer, and the second random sequence is positioned at the 5' end of the second primer,and the number of the first and second electrodes,the first specific sequence and the second specific sequence are respectively an upstream primer and a downstream primer aiming at a target sequence, and the first random sequence and the second random sequence are reversely complementary,the 5 'end base of the first primer and the 5' end base of the second primer are both connected with a fluorescent group, and the bases which are away from the 5 'end base of the first primer and the 5' end base of the second primer by a certain length are both connected with a quenching group.
- The pair of primers for quantitative PCR amplification according to claim 1, wherein the first and second specific sequences have a TM value of 55-65 ℃ and the first and second primers have a TM value of 65-75 ℃.
- The pair of primers for quantitative PCR amplification according to claim 1, wherein the first random sequence and the second random sequence have a length of 16-30bp, and the first specific sequence and the second specific sequence have a length of 16-30 bp.
- The pair of primers for quantitative PCR amplification according to claim 1, wherein the 2 nd to 5 th bases at the 5 'ends of the first and second primers are modified by thio, and the 1 st to 5 th bases at the 3' ends of the first and second primers are modified by thio.
- The pair of primers for quantitative PCR amplification according to claim 4, wherein the thio-modification is any one selected from the group consisting of a phosphorothioate-type modification, a methylsulfate-type modification and a peptide nucleic acid modification.
- The pair of primers for quantitative PCR amplification according to claim 1, wherein at least one of the first primer and the second primer has a 5' end modified by phosphorylation.
- The pair of primers for quantitative PCR amplification according to claim 1, wherein the quencher group is linked to a base 10 to 30bp away from the 5 'terminal base of the first primer and the 5' terminal base of the second primer.
- A quantitative PCR amplification kit comprising the pair of quantitative PCR amplification primers according to any one of claims 1 to 7.
- A quantitative PCR amplification method, wherein the quantitative PCR amplification is performed using the quantitative PCR amplification primer set according to any one of claims 1 to 7 or the quantitative PCR amplification kit according to claim 8.
- The method of claim 9, wherein the method comprises two rounds of amplification comprising:carrying out first round linear amplification on the quantitative PCR amplification primer pair and the template at an annealing temperature of 55-65 ℃; andand carrying out second round of circular amplification on the products of the first round of linear amplification at the annealing temperature of 65-72 ℃.
- The method of claim 10, wherein the amplification reaction sequence of the method is as follows:
- a method for quantitative analysis of a DNA sample to be tested, comprising:the method according to any one of claims 9 to 11, wherein the DNA sample to be tested is subjected to fluorescent quantitative PCR amplification and the quantitative analysis is carried out based on the collected fluorescent signal.
- A method for performing gene expression differential analysis of a specific gene in a plurality of test DNA samples, wherein each of the plurality of test DNA samples comprises a cDNA sequence of the specific gene, the method comprising:the method according to any one of claims 9 to 11, wherein the plurality of DNA samples to be tested are subjected to fluorescent quantitative PCR amplification respectively, and quantitative analysis is realized based on the collected fluorescent signals; andcomparing the quantitative analysis results of the plurality of DNA samples to be tested so as to determine the gene expression difference of the specific genes of the plurality of DNA samples to be tested.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2017/089197 WO2018232596A1 (en) | 2017-06-20 | 2017-06-20 | Quantitative pcr amplification primer pair and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110603328A true CN110603328A (en) | 2019-12-20 |
| CN110603328B CN110603328B (en) | 2024-01-12 |
Family
ID=64736263
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201780090496.8A Active CN110603328B (en) | 2017-06-20 | 2017-06-20 | Quantitative PCR amplification primer pair and application thereof |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN110603328B (en) |
| WO (1) | WO2018232596A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114875116A (en) * | 2022-04-27 | 2022-08-09 | 广州博懿瑞生物科技有限公司 | Self-quenching fluorescence primer and design method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2512631A (en) * | 2013-04-03 | 2014-10-08 | Rupert Maxwell Gaut | Quantitative detection of specific nucleic acid sequences |
| CN104404142A (en) * | 2014-11-11 | 2015-03-11 | 中国科学院上海微***与信息技术研究所 | Fluorescent probe for fluorescent quantitative PCR reactions |
| US20160237472A1 (en) * | 2012-11-30 | 2016-08-18 | Beijing Tag-Array Molecular Test Co., Ltd | Primer middle sequence interference pcr technology |
| US20160369327A1 (en) * | 2013-06-18 | 2016-12-22 | Lgc Limited | Fluorophore-based oligonucleotide probes with a universal element |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755379B (en) * | 2016-12-13 | 2021-06-04 | 海南医学院 | Dimer mutation fluorescent primer quantitative PCR method for synchronously quantifying and genotyping 4 aspergillus |
-
2017
- 2017-06-20 WO PCT/CN2017/089197 patent/WO2018232596A1/en active Application Filing
- 2017-06-20 CN CN201780090496.8A patent/CN110603328B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160237472A1 (en) * | 2012-11-30 | 2016-08-18 | Beijing Tag-Array Molecular Test Co., Ltd | Primer middle sequence interference pcr technology |
| GB2512631A (en) * | 2013-04-03 | 2014-10-08 | Rupert Maxwell Gaut | Quantitative detection of specific nucleic acid sequences |
| US20160369327A1 (en) * | 2013-06-18 | 2016-12-22 | Lgc Limited | Fluorophore-based oligonucleotide probes with a universal element |
| CN104404142A (en) * | 2014-11-11 | 2015-03-11 | 中国科学院上海微***与信息技术研究所 | Fluorescent probe for fluorescent quantitative PCR reactions |
Non-Patent Citations (4)
| Title |
|---|
| HUANTING LIU 等: "Placental exosomes in normal and complicated pregnancy", BMC BIOTECHNOL ., pages 130 - 134 * |
| JURATE BITINAITE等: "USER™ friendly DNA engineering and cloning method by uracil excision", NUCLEIC ACIDS RES, vol. 35, no. 6, pages 1992 - 2002, XP055170276, DOI: 10.1093/nar/gkm041 * |
| 郭秋平等: "用荧光双链引物特异扩增并定量核酸", 《厦门大学学报(自然科学版)》 * |
| 郭秋平等: "用荧光双链引物特异扩增并定量核酸", 《厦门大学学报(自然科学版)》, vol. 41, no. 01, 10 February 2002 (2002-02-10), pages 108 - 111 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114875116A (en) * | 2022-04-27 | 2022-08-09 | 广州博懿瑞生物科技有限公司 | Self-quenching fluorescence primer and design method and application thereof |
| CN114875116B (en) * | 2022-04-27 | 2023-08-29 | 广州博懿瑞生物科技有限公司 | Self-quenching fluorescence primer and design method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2018232596A1 (en) | 2018-12-27 |
| CN110603328B (en) | 2024-01-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6571895B1 (en) | Nucleic acid probe and genomic fragment detection method | |
| EP2534263B1 (en) | Methods and compositions for universal detection of nucleic acids | |
| US20210230670A1 (en) | Solid phase nucleic acid target capture and replication using strand displacing polymerases | |
| SG172965A1 (en) | Single-cell nucleic acid analysis | |
| JP3844996B2 (en) | Melting curve analysis method for repetitive PCR products | |
| CN110878356A (en) | Multiplex nucleic acid index amplification probe and tumor multi-target detection application thereof | |
| US20180237853A1 (en) | Methods, Compositions and Kits for Detection of Mutant Variants of Target Genes | |
| CN112852927A (en) | Isothermal amplification system and method based on fluorescence self-inhibition probe | |
| US8642264B2 (en) | Method of quantifying target and reference nucleic acid | |
| EP4083229A1 (en) | Methods and compositions for nucleic acid detection | |
| US20200407794A1 (en) | Molecular targets for fetal nucleic acid analysis | |
| CN110603328B (en) | Quantitative PCR amplification primer pair and application thereof | |
| EP3447145B1 (en) | Target nucleic acid sequence detection method using multiple amplification nested signal amplification | |
| EP3377652B1 (en) | A method and a kit for nucleic acid detection | |
| CN110612355B (en) | Composition for quantitative PCR amplification and application thereof | |
| JP2024515076A (en) | Amplification of single-stranded DNA | |
| US20090305288A1 (en) | Methods for amplifying nucleic acids and for analyzing nucleic acids therewith | |
| WO2024063036A1 (en) | Method for detecting methylation of genome dna | |
| JP2020516274A (en) | Library quantification and qualification | |
| WO2023067110A1 (en) | Methods and compositions for detection of mutant nucleic acid sequences | |
| EP3377651B1 (en) | A method and a kit for nucleic acid detection | |
| Andersen | Optimal Design of PCR Assays for Circulating DNA | |
| CN116334185A (en) | Nucleic acid detection kit based on PER-Cas12a | |
| JP2024523655A (en) | Methods and compositions for nucleic acid analysis | |
| WO2024015999A1 (en) | Methods, systems and compositions for detection of multiple analytes |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |