WO2018232597A1 - 用于定量pcr扩增的组合物及其应用 - Google Patents

用于定量pcr扩增的组合物及其应用 Download PDF

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WO2018232597A1
WO2018232597A1 PCT/CN2017/089198 CN2017089198W WO2018232597A1 WO 2018232597 A1 WO2018232597 A1 WO 2018232597A1 CN 2017089198 W CN2017089198 W CN 2017089198W WO 2018232597 A1 WO2018232597 A1 WO 2018232597A1
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Prior art keywords
primer
sequence
specific
amplification
pcr amplification
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English (en)
French (fr)
Inventor
杨林
张薇婷
高雅
张艳艳
陈芳
夏军
蒋慧
徐讯
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MGI Tech Co Ltd
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MGI Tech Co Ltd
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Priority to PCT/CN2017/089198 priority Critical patent/WO2018232597A1/zh
Priority to CN201780090491.5A priority patent/CN110612355B/zh
Publication of WO2018232597A1 publication Critical patent/WO2018232597A1/zh
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    • 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
    • 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

Definitions

  • the present invention relates to the field of biotechnology, and in particular to quantitative PCR amplification, and more particularly to compositions for quantitative PCR amplification and uses thereof.
  • Real-time PCR is the quantitative and qualitative analysis of the starting template by real-time detection of the fluorescence signal of each cycle product in the PCR amplification reaction.
  • a fluorescent chemical substance was introduced.
  • the PCR reaction product accumulated continuously, and the fluorescence signal intensity also increased in proportion.
  • a fluorescence intensity signal is collected every cycle, so that the change in the amount of the product can be monitored by the change in fluorescence intensity to obtain a fluorescence amplification curve.
  • Real-time PCR is a leap in DNA quantification technology. Using this technology, quantitative and qualitative analysis of DNA and RNA samples can be performed.
  • the present invention aims to solve at least one of the technical problems existing in the prior art. To this end, it is an object of the present invention to provide a real-time fluorescent quantitative PCR technique with high amplification efficiency and specificity, and a corresponding PCR primer pair and probe composition.
  • the most common method for qPCR is the dye method relying on SYBR and the probe method relying on Taqman probe; the former uses fluorescent dye to indicate the increase of amplification, and the latter uses the probe which specifically hybridizes with the target sequence to indicate amplification.
  • the increase in product in which the TaqMan probe method is currently used more.
  • the TaqMan probe is an oligonucleotide probe whose fluorescence is associated with amplification of the sequence of interest. It is designed to sequence pair with 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.
  • the fluorescence emitted by the fluorophore is quenched by the proximity of the quencher group at the 3' end.
  • the 5' exonuclease activity of the polymerase cleaves the probe such that the fluorophore is separated from the quenching group.
  • the fluorescence intensity is proportional to the amount of amplified product.
  • the design of the PCR primer pair and the corresponding probe cannot be ignored, and the design of the PCR primer pair is particularly important.
  • PrimerDesigner210 (Scientific and Educational Software) is widely used for its shortness and functionality.
  • PCR specificity and amplification efficiency depend too much on the design of the primer, in some repeat regions, high GC regions or advanced structures. Regional primers are often difficult to obtain good results, and designing primers and primer optimization requires a lot of effort and material resources. Therefore, the inventors conducted a series of design and experimental explorations to improve the problem. Moreover, the inventors have unexpectedly discovered that a complementary sequence is added to the 5' end of a pair of conventional primers to form a primer with a stable primer-dimer structure having a "5'-end inverted complement and a 3'-end overhang". Yes, it can effectively improve the above problems.
  • the inventors have found that the above-mentioned primer pair having a stable primer-dimer structure having a "5'-end reverse complementation, a 3'-end overhang" and a corresponding specific probe are subjected to qPCR, and the specificity is strong and expanded. High efficiency, accurate and reliable when used for quantitative analysis of DNA or RNA and differential analysis of gene expression. Furthermore, the inventors proposed a qPCR technique combining a specific PCR primer with a Taqman probe to design a pair of special primers and a specific probe for the target region, and the technique relies on the specially designed primers.
  • the qPCR technology has the characteristics of high specificity and high amplification efficiency compared with the conventional qPCR, and has very good homogeneity in qPCR multiplex amplification, and has certain advantages for multi-site qPCR detection.
  • the invention provides a composition for quantitative PCR amplification.
  • the composition for quantitative PCR amplification comprises: a pair of PCR primer pairs and a specific probe, the PCR primer pair comprising a first primer and a second primer, wherein the a primer comprising a first specific sequence located at the 3' end of the first primer and a first random sequence, the first random sequence being located at the 5' end of the first primer,
  • the second primer comprises a second specific sequence located at the 3' end of the second primer and a second random sequence located at the 5' end of the second primer
  • the said specific sequence and the second specific sequence are respectively an upstream primer and a downstream primer for the target sequence, and the first random sequence and the second random sequence are inversely complementary, 5' of the specific probe A fluorophore is attached to the end, and a quenching group is attached to the 3' end, and the sequence of the specific probe is complementary to the sequence between the upstream primer and the downstream primer of
  • the PCR primer pair is effective in reducing GC bias during PCR amplification and increasing amplification specificity.
  • the PCR enrichment process of the second generation sequencing library with conventional primers brings about a certain degree of GC bias, and the PCR primer pair of the present invention (sometimes referred to as "locking primer”) can effectively reduce the process of library PCR enrichment.
  • GC bias when qPCR is carried out using a composition comprising the PCR primer pair and the corresponding specific probe, it is more specific and has higher amplification efficiency than conventional qPCR, and is used for quantitative analysis and gene expression of DNA or RNA. When the difference is analyzed, the results are more accurate and reliable.
  • the composition of the present invention has very good homogeneity for qPCR multiplex amplification, and has certain advantages for multi-site qPCR detection.
  • the invention provides a quantitative PCR amplification kit.
  • the kit comprises the composition described above for quantitative PCR amplification.
  • the specificity is stronger and the amplification efficiency is higher than that of the conventional qPCR, and when used for quantitative analysis of DNA or RNA and differential analysis of gene expression, The result is more accurate and reliable.
  • the composition of the present invention has very good homogeneity for qPCR multiplex amplification, and has certain advantages for multi-site qPCR detection.
  • the invention provides a quantitative PCR amplification method.
  • the method performs the quantitative PCR amplification using a composition for quantitative PCR amplification or a quantitative PCR amplification kit as described above.
  • quantitative PCR amplification of the template can be effectively achieved by this method.
  • the method can increase the specificity of quantitative PCR amplification, effectively reduce the production of non-specific products, and improve the amplification efficiency, thereby improving the accuracy of the quantitative results.
  • the invention provides a method for quantitative analysis of a sample of DNA to be tested.
  • the method comprises: performing quantitative PCR amplification on a DNA sample to be tested according to the quantitative PCR amplification method described above, and performing quantitative analysis based on the collected fluorescence signal.
  • the specificity of PCR amplification is good, the amplification efficiency is high, and the quantitative analysis results are accurate and reliable.
  • the present invention provides a method for performing differential gene expression analysis of a specific gene on a plurality of DNA samples to be tested.
  • the plurality of DNA samples to be tested each comprise a cDNA sequence of the specific gene, the method comprising: respectively, the plurality of DNAs to be tested according to the quantitative PCR amplification method described above
  • the sample is subjected to real-time PCR amplification, and quantitative analysis is performed based on the collected fluorescent signal; and the quantitative analysis results of the plurality of DNA samples to be tested are compared to determine gene expression differences of specific genes of the plurality of DNA samples to be tested.
  • the results of gene expression difference analysis are accurate and reliable.
  • composition for quantitative PCR amplification comprising a pair of PCR primer pairs and a specific probe of the present invention and use thereof have at least one of the following advantages:
  • the design strategy of the PCR primer pair of the invention simplifies the primer design flow and optimizes the experimental steps.
  • the composition of the primer pair consists of a specific sequence at the 3' end and a random sequence at the 5' end (complementary sequence), and the forward and reverse primers.
  • the formation of a stable dimer structure by complementary sequences does not require the strict conditions of conventional primers, greatly simplifying the design process.
  • the PCR primer does not require special optimization at the 5' end to achieve a good amplification effect, and the primer design time is short.
  • the design of the PCR primer pair of the present invention does not need to consider these problems, because the structure of the locked primer itself is a complementary stable dimer at the 5' end and the 3' end of the dimer can also be normal and specific sequence. Complementary extension occurs, whereas if the conventional primer forms a dimeric structure at the 5' end, there is not enough sequence and specific complementary sequence for binding at the 3' end.
  • the potential energy of the complementary sequence of the 5' end between the two primers of the locked primer of the present invention is much larger than that of the self-palindrome, so that even if the complementary sequence is present at the 3' end and the 5' end, the 5' end dimerization is preferentially formed.
  • Body structure is much larger than that of the self-palindrome, so that even if the complementary sequence is present at the 3' end and the 5' end, the 5' end dimerization is preferentially formed.
  • PCR amplification using the primer pair of the present invention can increase the specificity of PCR amplification and effectively reduce the production of non-specific products; starting from the second cycle of PCR, the 5' base of the primer (random sequence) and The 5' end of the newly generated template is reversely complementary, and the 3' end specific sequence of the primer and the newly generated 3' end of the template are reversely complementary, that is, the primer and template binding recognition sites are 2 (As in Figure 2), thereby, the binding ability of the primer and the template, as well as the specificity of amplification, are significantly increased. The binding rate of the primer and the template is improved, and the amplification efficiency is also effectively improved.
  • PCR amplification using the PCR primer pair of the present invention can effectively reduce the GC bias of different templates in the amplification of the sequencing library (especially the second generation sequencing library), because the PCR amplification can only be combined after the template is denatured. An effective amplification occurs.
  • GC preference arises because in some high GC regions, the template refolds rapidly during PCR, and the template is renatured without the primers binding, resulting in the inability of these regions to be efficiently amplified.
  • the locking primer and the template of the present invention have two binding sites, which can greatly improve the binding ability with the template, thereby effectively combining the high GC templates, thereby reducing the GC bias.
  • the product obtained by the quantitative PCR amplification method of the present invention is a notched ring-like substance (that is, the 5' end and the 3' end of the ring type are not linked), and an experiment for cyclization operation is required for some products.
  • cyclization can be achieved by simply adding a ligase, which does not require complicated denaturation and quenching processes, and can effectively simplify the experimental process.
  • the quantitative PCR amplification method of the present invention relies on a specially designed PCR primer pair in a composition for quantitative PCR amplification.
  • the qPCR using the composition of the invention has higher specificity and higher amplification efficiency, and is more accurate and reliable when used for quantitative analysis of DNA or RNA and differential expression of gene expression;
  • the composition of the invention has very good homogeneity for qPCR multiplex amplification and has certain advantages for multi-site qPCR detection.
  • composition for quantitative PCR amplification, the quantitative PCR amplification kit and the quantitative PCR amplification method of the invention can be widely used for quantitative analysis of DNA or RNA, gene expression difference analysis, genotyping and pathogen detection. And other fields.
  • FIG. 1 shows a schematic structural view of a PCR primer pair (ie, a locked primer) of the present invention, in accordance with one embodiment of the present invention
  • FIG. 2 is a schematic diagram showing the binding of a primer to a newly generated strand in the circular amplification of the present invention according to an embodiment of the present invention
  • FIG. 3 shows a schematic flow diagram of quantitative PCR amplification using the composition of the present invention, in accordance with an embodiment of the present invention
  • Figure 4 is a graph showing the results of agarose gel electrophoresis detection of the PCR amplification product of the conventional SRY primer in Example 1.
  • Figure 5 shows the PP primer amplification fluorescence detection signal in Example 1
  • Figure 6 shows a conventional primer amplification fluorescence detection signal in Example 1
  • Figure 7 is a graph showing the standard value of the CT value of the PP primer in Example 1;
  • Figure 8 is a graph showing the standard curve of conventional primer CT values in Example 1.
  • Fig. 9 shows the results of qPCR fluorescence signal detection of each sample in Example 2.
  • first and second are used for descriptive purposes only, and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, features defining “first” and “second” may include one or more of the features either explicitly or implicitly. Further, in the description of the present invention, the meaning of "a plurality" is two or more unless otherwise specified.
  • the invention provides a composition for quantitative PCR amplification.
  • the composition for quantitative PCR amplification comprises: a pair of PCR primer pairs and a specific probe, the PCR primer pair comprising a first primer and a second primer, wherein the a primer comprising a first specific sequence located at the 3' end of the first primer and a first random sequence, the first random sequence being located at the 5' end of the first primer,
  • the second primer comprises a second specific sequence located at the 3' end of the second primer and a second random sequence located 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 for the target sequence, and the first random sequence and the second random sequence are inversely complementary
  • the specific The 5' end of the sex probe is linked to a fluorescent group, and the 3' end is linked to a quenching group, and the sequence of the specific probe is complementary to the sequence between the upstream primer and the downstream primer of the
  • the PCR primer pair is effective in reducing GC bias during PCR amplification and increasing amplification specificity.
  • the PCR enrichment process of the second generation sequencing library with conventional primers brings about a certain degree of GC bias, and the PCR primer pair of the present invention (sometimes referred to as "locking primer”) can effectively reduce the process of library PCR enrichment.
  • GC bias when qPCR is carried out using a composition comprising the PCR primer pair and the corresponding specific probe, it is more specific and has higher amplification efficiency than conventional qPCR, and is used for quantitative analysis and gene expression of DNA or RNA. When the difference is analyzed, the results are more accurate and reliable.
  • the composition of the present invention has very good homogeneity for qPCR multiplex amplification, and has certain advantages for multi-site qPCR detection.
  • first random sequence and the “second random sequence” of the present invention may be a random sequence or a fixed sequence, as long as the two are complementary to each other.
  • the first specific sequence and the second specific sequence have a TM value of 55-65 degrees Celsius
  • the first primer and the second primer have a TM value of 65-75 degrees Celsius.
  • the PCR can be subjected to a linear amplification of the first round of low annealing temperature (55-65 degrees Celsius), and then a high annealing temperature (65-72 degrees Celsius) is used in the subsequent cycle - that is, the second round Cyclic amplification. Due to the high annealing temperature during the circular amplification process, the specific sequence cannot bind to the specific site alone (the specific sequence has a TM value of only 55-65 degrees Celsius), only when the 5' end of the locked primer binds. To the 5' end of the template, and the 3' end of the locked primer binds to the specific site of the template, the PCR can be effectively amplified, that is, the round amplification is actually a double-binding site. increase.
  • the PCR primer pair of the invention is suitable for PCR amplification and library construction for any form of DNA sample to be tested.
  • sample of DNA to be tested described in the present invention is somewhat different from the conventional understanding, and it is conventionally understood that the treated DNA is not included.
  • the "sample of DNA to be tested” may include treated DNA as well as untreated DNA (in the case of constructing a sequencing library, the genomic DNA of the sample is generally interrupted and sequenced for processing to obtain a carrier.
  • the DNA fragment of the corresponding platform is sequenced, and after subsequent amplification and other steps, the obtained product can be used for sequencing, and the DNA fragment carrying the corresponding platform sequencing linker is a "treated DNA fragment", correspondingly, after the above Treated, that is, "untreated DNA”). If it is directed to untreated DNA, amplification using the PCR primer pair of the present invention is to amplify a specific target fragment; if it is directed to the treated DNA, the amplified target fragment can be a DNA fragment of the entire genome.
  • the DNA sample to be tested is a processed DNA fragment carrying a universal sequence (eg, a sequencing linker)
  • the “universal sequence” herein is a sequence for pairing with a specific sequence in the primer, including sequencing.
  • the platform adaptor sequence ie, the sequencing adaptor
  • the first specific sequence and the second specific sequence must be capable of specifically recognizing the target sequence carrying the universal sequence, in other words, the target sequence at this time
  • "general sequence + target region sequence” when the DNA sample to be tested (that is, the PCR reaction template) is a DNA fragment that does not carry a universal sequence, correspondingly, the first specific sequence and the second specificity The sequence can specifically recognize the target sequence.
  • a sequencing linker sequence (ie, 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 as to make the PCR amplification product
  • the sequencing linker is ligated to enable efficient use in the sequencing platform.
  • At least one of the first primer and the second primer further comprises a tag sequence, whereby PCR amplification of a plurality of samples can be performed simultaneously, and based on the tag sequence pair Each sample is distinguished.
  • the position of the tag sequence in the first primer and the second primer is not particularly limited as long as it can function to distinguish each sample without affecting PCR amplification.
  • the tag sequence may be located between a specific sequence and a random sequence, whereby a tag sequence may be placed between the first specific sequence of the first primer and the first random sequence, and Or, a tag sequence is placed between the second specific sequence of the second primer and the second random sequence.
  • the tag sequence may also be arranged to be included in a random sequence, ie part of a random sequence.
  • the first random sequence and the second random sequence are 16-30 bp in length
  • the first specific sequence and the second specific sequence are 16-30 bp in length
  • the first primer and the second primer and the 1-5th base of the 3' end are thio-modified. Thereby, the cleavage of the enzyme can be effectively prevented.
  • 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-enzymed (for example, having 5'-3' or 3'-5' Degradation of Dicer Activity).
  • the thio modification is any one selected from the group consisting of a phosphorothioate type modification, a methyl sulfate type modification, and a peptide nucleic acid modification.
  • the 5' end of at least one of the first primer and the second primer is phosphorylated.
  • the nicked loop-like ring obtained by two rounds of amplification ie, the 5' end and the 3' end of the loop-like substance are not linked
  • a ligase can be joined by a ligase to form a complete loop.
  • DNA That is, a circular DNA library can be prepared based on the product of qPCR.
  • the design strategy of the PCR primer pair of the present invention is: 5 pairs of a conventional primer (including a forward primer and a reverse primer). Adding a complementary sequence to the end, the complementary sequence can be a random sequence or a fixed sequence. Thus, a pair of primers for PCR is designed to be 5'-end complementary, and the 3' end is prominent.
  • the lock primer (PadlockPrimer, PP) forms a stable primer-dimer structure between the primer pairs of the locked primer.
  • the length of the entire primer pair is 32-60 bp, and the TM value is higher (the TM values of the first primer and the second primer are generally 65-75 degrees Celsius), wherein the complementary sequence of the 5' end of the primer is locked (ie, the first random number
  • the sequence and the second random sequence are 16-30 bp in length, and the sequence may be random or fixed; the 3' end (ie, the first specific sequence and the second specific sequence) is 16-30 bp in length, and
  • the target sequences of the template are complementary and the TM value is low (typically 55-65 degrees Celsius).
  • the PCR primer pair of the present invention needs to perform PCR amplification by two different amplification sections (ie, two rounds of amplification) (refer to FIG. 3): in the first The amplification portion has an annealing temperature of 55-65 ° C and a cycle number of 1; in the second amplification portion, the annealing temperature is 65-72 ° C, and the number of cycles is 40-50. In the first amplification section, the PCR primer pair can only bind through the specific sequence at the 3' end and the template, so the annealing temperature of the cycle is low; in the second amplification portion, the PCR primer pair first passes through the 5' end.
  • the complementary sequence ie, the first random sequence and the second random sequence
  • binds to the newly generated template ie, the product of the first round of amplification
  • passes through the specific sequence at the 3' end ie, the first specific sequence and the second
  • the specific sequence is combined with the newly generated template, that is, the primer and template binding recognition sites are two, and the combination of the two anchor sites greatly increases the annealing temperature of the primer, so the annealing temperature is higher.
  • the 5' end and the 3' end of the primer can be efficiently circularly amplified only when they are simultaneously combined with the newly generated template, thus, two recognition sites are The specificity of the PCR amplification is greatly improved, and the two binding sites greatly improve the binding ability of the primer and the template, and the amplification efficiency of the PCR is improved.
  • PCR amplification using the PCR primer pair of the present invention relative to conventional PCR primers can significantly increase the specificity of PCR amplification, effectively reduce the production of non-specific products, and reduce the GC bias during amplification.
  • the use of such primers in sequencing especially in second-generation sequencing libraries, can effectively reduce the genome-wide GC bias in library enrichment amplification.
  • PCR amplification using the PCR primer pair of the present invention can be applied to product cyclization, and the obtained product can be directly ligated to obtain a circular DNA.
  • no additional steps such as denaturation, quenching, and the like are required, and the product can be looped by adding a ligation reaction system, thereby greatly simplifying the loop formation process, thereby simplifying the preparation process of the circular DNA library.
  • the specific probe has a sequence length of 18-30 bp and a TM value of 70-80 degrees Celsius.
  • the invention provides a quantitative PCR amplification kit.
  • the kit comprises the composition described above for quantitative PCR amplification.
  • the specificity is stronger and the amplification efficiency is higher than that of the conventional qPCR, and when used for quantitative analysis of DNA or RNA and differential analysis of gene expression, The result is more accurate and reliable.
  • the composition of the present invention has very good homogeneity for qPCR multiplex amplification, and has certain advantages for multi-site qPCR detection.
  • the invention proposes the use of a composition and a kit comprising the same.
  • the invention provides a quantitative PCR amplification method.
  • the method performs the quantitative PCR amplification using a composition for quantitative PCR amplification or a quantitative PCR amplification kit as described above.
  • quantitative PCR amplification of the template can be effectively achieved by this method.
  • the method can increase the specificity of quantitative PCR amplification, effectively reduce the production of non-specific products, and improve the amplification efficiency, thereby improving the accuracy of the quantitative results.
  • the method comprises two rounds of amplification: performing a first round of linear amplification of the PCR primer pair and the template in the presence of a specific probe at an annealing temperature of 55-65 degrees Celsius And a second round of circular amplification of the first linearly amplified product at an annealing temperature of 65-72 degrees Celsius.
  • the base of the 5' end of the first primer and the second primer and the base of the 5' end of the newly generated template are reversely complementary
  • the 3'-end specific base of the first primer and the second primer and the base of the newly generated 3' end of the template are reversely complementary, that is, the primer and template binding recognition sites are 2 (as shown in FIG. 2), thereby It can increase the specificity of PCR amplification and effectively reduce the production of non-specific products.
  • the amplification reaction procedure of the method is as follows:
  • the GC bias during PCR amplification is low, the amplification specificity is high, and the amplification effect is good.
  • the invention provides a method for quantitative analysis of a sample of DNA to be tested.
  • the method comprises: performing quantitative PCR amplification on a DNA sample to be tested according to the quantitative PCR amplification method described above, and performing quantitative analysis based on the collected fluorescence signal.
  • the specificity of PCR amplification is good, the amplification efficiency is high, and the quantitative analysis results are accurate and reliable.
  • the present invention provides a method for performing differential gene expression analysis of a specific gene on a plurality of DNA samples to be tested.
  • the plurality of DNA samples to be tested each comprise a cDNA sequence of the specific gene, the method comprising: respectively, the plurality of DNAs to be tested according to the quantitative PCR amplification method described above
  • the sample is subjected to real-time PCR amplification, and quantitative analysis is performed based on the collected fluorescent signal; and the quantitative analysis results of the plurality of DNA samples to be tested are compared to determine gene expression differences of specific genes of the plurality of DNA samples to be tested.
  • the results of gene expression difference analysis are accurate and reliable.
  • the product obtained by the quantitative PCR amplification method of the present invention is a notched loop-like substance (ie, the 5' end and the 3' end of the loop are not linked), and an experiment for cyclization is required for some products.
  • cyclization can be achieved by simply adding a ligase, which does not require complicated denaturation and quenching processes, and can effectively simplify the experimental process.
  • the invention also provides a method of preparing a circular DNA library. According to an embodiment of the invention, the method comprises the steps of:
  • the circular DNA library when the 5' end of one of the first primer and the second primer is phosphorylated, the circular DNA library is a single-stranded circular DNA library, when the first primer and the When the 5' end of the second primer is phosphorylated, the circular DNA library is a double-stranded circular DNA library.
  • a single-stranded or double-stranded circular DNA library can be efficiently prepared by using the method, and the obtained single-stranded or double-stranded circular DNA library has a good library quality for DNA preservation or library sequencing. The effect is good.
  • the obtained library is of good quality.
  • linear DNA digestion is utilized to remove linear DNA.
  • a universal sequence is added to both ends of the DNA sample to be tested.
  • the expression "universal sequence” as used herein refers to a sequence used to pair with a specific sequence in a primer, including a sequencing platform linker sequence, ie, a sequencing linker.
  • a sequencing platform linker sequence ie, a sequencing linker.
  • the amplification system is as follows:
  • the reaction conditions are as follows:
  • the obtained PCR product was purified by XP Beads (Agencourt AMPure XP, A53880, USA), purified by agarose electrophoresis (see Figure 4), and the quality of the PCR product was ready for use.
  • the strip NTC is a negative control (water is a template), and the strips 1, 2 are products obtained by male DNA amplification, and the product size is 79 bp, and the test is qualified.
  • the product obtained in step 2 was diluted 10 times, 100 times, 1000 times, 10000 times and 100000 times in the ventilated place, and the obtained DNA was 10%, 1%, 0.1%, 0.01 of the original template concentration, respectively. %, 0.001%;
  • the gradient template obtained above was taken with PP primers (primers PP-SRY F and PP-SRY R, primer sequences are shown in Table 4 below, schematic diagram is shown in Figure 1) and conventional primers (primers SRY F and SRY R, the sequence is shown in the table below). 4) The qPCR reaction was carried out, and three reactions were repeated for each gradient. The two primers and their probe sequences are shown in Table 4.
  • the qPCR reaction was carried out using Takara's Premix Ex Taq TM (Cat. No. RR390A) kit.
  • the reaction system was as follows:
  • the qPCR reaction was carried out on ABI's Stepone, and the reaction conditions were as follows:
  • FIG. 1 The flow of qPCR amplification using the PP primer of the present invention is shown in FIG.
  • FIG. 5 is a PP primer amplification fluorescence detection signal
  • FIG. 6 is a conventional primer amplification fluorescence detection signal.
  • FIG. 7 shows the PP primer standard curve.
  • FIG. 8 shows a conventional primer standard curve.
  • Two pairs of locked primers ie, PP primers, schematic diagram of the primer structure shown in Figure 1
  • corresponding probes were designed on the specific genes SRY and DSY14 on the Y chromosome
  • a pair of locked primers and probes were designed on the autosomal GADPH gene.
  • the sequence of each of the locking primers and the corresponding probe is shown in Table 4.
  • the fluorescent group and the quenching group of the specific probe labeled on the Y chromosome are FAM and BHQ1, respectively
  • the fluorescent group and the quenching group labeled by the probe on the GADPH gene are HEX and BHQ1, respectively.
  • a multiplex multiplex PCR was performed on the primers to identify fetal-specific Y chromosomes in plasma.
  • the plasma of pregnant women with 6/8/10 weeks of gestational age was selected as 600 ul, and each of the pregnant women and men of different gestational weeks was extracted with Qiagen plasma free DNA extraction kit (QIAamp Circulating Nucleic Acid Kit, Cat. No./ID: 55114
  • Qiagen plasma free DNA extraction kit QIAamp Circulating Nucleic Acid Kit, Cat. No./ID: 55114
  • the obtained plasma free DNA was dissolved in 25 ul of AE, and a qPCR reaction was carried out using a PP primer, wherein the test was based on water.
  • FIG. 1 The flow of qPCR amplification using the PP primer of the present invention is shown in FIG.
  • reaction system a reaction product of Takara qPCR Premix Ex Taq TM (RR390A) kit, the reaction system was as follows:
  • Reverse primer mixture (10 ⁇ M): PP-SRY R, PP-DSY14R and PP-GADPH R mixture, each primer concentration was 10 ⁇ M;
  • Probe mixture SRY-Probe, DSY14-Probe and GADPH-Probe mixture, each probe concentration is ⁇ M)
  • the qPCR reaction was carried out on ABI's Stepone, and the reaction conditions were as follows:
  • HEX fluorescence can be detected in 6 samples, and the CT values are not much different, indicating that the cfDNA extraction is successful, and the total amount of cfDNA is not much different; no fluorescence is detected in water; among them, three male fetal plasma samples FAM fluorescence was detected, and the CT value decreased with the increase of gestational age. FAM fluorescence was not detected in the plasma samples of three female fetuses. This method can correctly detect fetal gender by plasma free DNA of pregnant women as low as 6 weeks.
  • the composition for quantitative PCR amplification of the present invention can be effectively used for quantitative PCR amplification of a DNA sample to be tested, and has higher specificity and higher amplification efficiency than conventional qPCR, and is used for DNA or RNA.
  • the quantitative analysis and differential analysis of gene expression are more accurate and reliable.
  • the composition of the present invention has very good homogeneity for qPCR multiplex amplification, and has certain advantages for multi-site qPCR detection.

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Abstract

提供了用于定量PCR扩增的组合物，其包括：一对PCR引物对和一条特异性探针，所述PCR引物对包括第一引物和第二引物，其中，所述第一引物包含第一特异性序列和第一随机序列，所述第一特异性序列位于所述第一引物的3'端，所述第一随机序列位于所述第一引物的5'端，所述第二引物包含第二特异性序列和第二随机序列，所述第二特异性序列位于所述第二引物的3'端，所述第二随机序列位于所述第二引物的5'端，并且，所述第一特异性序列和所述第二特异性序列分别为针对靶序列的上游引物和下游引物，所述第一随机序列和所述第二随机序列反向互补，所述特异性探针的序列与所述靶序列的上游引物和下游引物之间的序列互补配对。

Description

用于定量PCR扩增的组合物及其应用
优先权信息
无
技术领域
本发明涉及生物技术领域，具体而言，涉及定量PCR扩增，更具体地，涉及用于定量PCR扩增的组合物及其应用。
背景技术
实时荧光定量PCR是通过对PCR扩增反应中每一个循环产物荧光信号的实时检测从而实现对起始模板定量及定性的分析。在实时荧光定量PCR反应中，引入了一种荧光化学物质，随着PCR反应的进行，PCR反应产物不断累计，荧光信号强度也等比例增加。每经过一个循环，收集一个荧光强度信号，这样就可以通过荧光强度变化监测产物量的变化，从而得到一条荧光扩增曲线图。
实时荧光定量PCR技术是DNA定量技术的一次飞跃。运用该项技术，可以对DNA、RNA样品进行定量和定性分析。
然而，目前的实时荧光定量PCR技术仍有待改进。
发明内容
本发明旨在至少解决现有技术中存在的技术问题之一。为此，本发明的一个目的在于提出一种扩增效率高、特异性好的实时荧光定量PCR技术及相应的PCR引物对和探针组合物。
首先，需要说明的是，本发明是基于发明人的下列发现和工作而完成的：
目前qPCR最常用的方法是依赖于SYBR的染料法和依赖于Taqman探针的探针法；前者利用荧光染料来指示扩增的增加，后者利用与靶序列特异杂交的探针来指示扩增产物的增加，其中，TaqMan探针法是目前使用较多的技术。TaqMan探针是一种寡核苷酸探针，它的荧光与目的序列的扩增相关。它设计为与目标序列上游引物和下游引物之间的序列配对。荧光基团连接在探针的5’末端，而淬灭剂则在3’末端。当完整的探针与目标序列配对时，荧光基团发射的荧光因与3’端的淬灭基团接近而被淬灭。但在进行延伸反应时，聚合酶的5’外切酶活性将探针进行酶切，使得荧光基团与淬灭基团分离。随着扩增循环数的增加，释放出来的荧光基团不断积累。因此荧光强度与扩增产物的数量呈正比关系。
该方法虽然特异性强，但需要设计特异性的探针，引物和探针需要进行优化，有些复杂的区域很难得到一个比较好的引物和探针组合。在多重检测中，探针法更难去优化一个较好的引物组合(引物对和特异性探针)，并且在某些区域虽然荧光检测是特异的，但是实际上还是会经常遇到有非特异的PCR产物出现的情况，虽然这部分产物不会产生荧光， 但会影响PCR引物对的扩增效率和不同扩增区域的均一性，在一些相对定量中会产生较大的偏差。
因而，利用TaqMan探针法进行qPCR时，采用的PCR引物对及对应的探针的设计不容忽视，而其中的PCR引物对的设计尤为重要。
而针对其中的PCR引物对的设计，网上有许多提供免费在线引物设计服务的网址和软件，例如：NetPrimer(www.premierbiosoft.com)。常用的单机引物设计软件有很多不同的产品，各有其优点。RightprimerTM(Bio2Disk)具有超强校对功能，可将待扩增序列的可能引物在很短的时间内,通过查找Genebank，而与背景DNA校对，从而找到特异性极佳之引物。OligoTM(Molecular Biology Insights,Inc.)适合于MultiplexPCR和ConsensusPCR的引物设计，并能给出适合的PCR条件。PrimerPremier(或者Premierbiosoft)，可以在核酸序列未知的情况下，通过蛋白质序列来设计引物，这一功能在仅知道部分蛋白质序列，而又想克隆新基因时特别有用。PrimerDesigner210(Scientific and Educational Software)以其短小精悍，功能齐全而得到广泛的应用。
但在实际中工作中，常规引物设计软件设计的好的引物不一定会产生好的结果，特别是那些高GC区域以及模板中有和其他目的区域相似的序列，最终导致引物扩增得到的产物特异性不强，PCR扩增效率不高，而去优化这些区域的引物又往往难以得到满意的结果。
发明人研究后认为，当前的PCR引物设计需要严格按照引物设计条件进行设计，PCR特异性和扩增效率过多依赖于引物设计的好坏，在一些重复区域、高GC区域或是有高级结构的区域引物往往难以得到好的效果，设计引物和引物优化需要花费大量的精力和物力。因而，发明人进行了一系列设计和实验探索，以期改善这一问题。并且，发明人意外地发现：在一对常规引物的5’端加上一段互补的序列，形成的具有“5’端反向互补，3’端突出”的稳定的引物二聚体结构的引物对，能够有效改善上述问题。
进而，发明人发现，利用上述的具有“5’端反向互补，3’端突出”的稳定的引物二聚体结构的引物对，以及对应的特异性探针进行qPCR，特异性强、扩增效率高，用于DNA或RNA的定量分析、基因表达差异分析时，结果精确、可靠。进而，发明人提出了一种特殊PCR引物和Taqman探针相结合的qPCR技术，对目的区域设计一对特殊的引物和一条特异性探针，该技术就依赖于这对特殊设计的引物。该qPCR技术相对于常规的qPCR具有特异性强、扩增效率高等特点，并且在qPCR多重扩增中具有非常好的均一性，对多位点qPCR检测具有一定的优势。
因而，在本发明的第一方面，本发明提供了一种用于定量PCR扩增的组合物。根据本发明的实施例，该用于定量PCR扩增的组合物包括：一对PCR引物对和一条特异性探针，所述PCR引物对包括第一引物和第二引物，其中，所述第一引物包含第一特异性序列和第一随机序列，所述第一特异性序列位于所述第一引物的3’端，所述第一随机序列位于所述第一引物的5’端，所述第二引物包含第二特异性序列和第二随机序列，所述第二特异性序列位于所述第二引物的3’端，所述第二随机序列位于所述第二引物的5’端，并且，所述第 一特异性序列和所述第二特异性序列分别为针对靶序列的上游引物和下游引物，所述第一随机序列和所述第二随机序列反向互补，所述特异性探针的5’末端连接有荧光基团，3’末端连接有淬灭基团，且所述特异性探针的序列与所述靶序列的上游引物和下游引物之间的序列互补配对。发明人惊奇地发现，本发明的组合物中，该PCR引物对能有效降低PCR扩增过程中的GC偏向性，提高扩增特异性。具体地，用常规引物在二代测序文库PCR富集过程会带来一定的GC偏向性，而本发明的PCR引物对(有时也称为“锁定引物”)能有效降低文库PCR富集过程中的GC偏向性。进而，利用包含该PCR引物对及对应的特异性探针的组合物进行qPCR时，相对于常规的qPCR，特异性更强、扩增效率更高，用于DNA或RNA的定量分析、基因表达差异分析时，结果更加精确、可靠。并且本发明的组合物用于qPCR多重扩增时具有非常好的均一性，对多位点qPCR检测具有一定的优势。
在本发明的第二方面，本发明提供了一种定量PCR扩增试剂盒。根据本发明的实施例，该试剂盒包含前面所述的用于定量PCR扩增的组合物。根据本发明的实施例，相对于常规的qPCR，利用包含本发明的组合物进行qPCR时，特异性更强、扩增效率更高，用于DNA或RNA的定量分析、基因表达差异分析时，结果更加精确、可靠。并且本发明的组合物用于qPCR多重扩增时具有非常好的均一性，对多位点qPCR检测具有一定的优势。
在本发明的第三方面，本发明提供了一种定量PCR扩增方法。根据本发明的实施例，该方法利用前面所述的用于定量PCR扩增的组合物或者定量PCR扩增试剂盒进行所述定量PCR扩增。由此，利用该方法能够有效实现模板的定量PCR扩增。并且该方法能够增加定量PCR扩增的特异性，有效降低非特异性产物的产生，并提高扩增效率，进而能够提高定量结果的准确度。
在本发明的第四方面，本发明提供了一种对待测DNA样品进行定量分析的方法。根据本发明的实施例，该方法包括：根据前面所述的定量PCR扩增方法，将待测DNA样品进行荧光定量PCR扩增，并基于采集的荧光信号实现定量分析。由此，PCR扩增的特异性好，扩增效率高，定量分析结果准确可靠。
在本发明的第五方面，本发明提供了一种对多个待测DNA样品进行特定基因的基因表达差异分析的方法。根据本发明的实施例，所述多个待测DNA样品均包含所述特定基因的cDNA序列，所述方法包括：根据前面所述的定量PCR扩增方法，分别将所述多个待测DNA样品进行荧光定量PCR扩增，并基于采集的荧光信号实现定量分析；以及比较多个待测DNA样品的定量分析结果，以便确定多个待测DNA样品的特定基因的基因表达差异。由此，基因表达差异分析的结果准确可靠。
根据本发明的实施例，本发明的包含一对PCR引物对和一条特异性探针的用于定量PCR扩增的组合物及其应用具有下列优点的至少之一：
1、本发明的PCR引物对的设计策略，简化了引物设计流程、优化了实验步骤，引物对的组成由3’端的特异性序列和5’端的随机序列(互补序列)组成，正反向引物通过互补序列形成一个稳定的二聚体结构，不需要满足常规引物严格的条件，大大简化了设计流程。该PCR引物对5’端不需要特殊的优化就能达到一个很好的扩增效果，引物设计时间短。在 常规引物设计过程中需要避免引物5’端和5’端的互补、引物自身形成回文结构等，以保证引物之间不能形成二聚体结构和发生自身延伸进行PCR。但本发明的PCR引物对的设计不需要考虑这些问题，因为锁定引物自身的结构就是5’端互补稳定的二聚体且此时的二聚体的3’端还能正常的和特异性序列进行互补发生延伸，而常规引物如果5’端形成二聚体结构，3’端就没有足够的序列和特异性互补序列进行结合。并且，本发明的锁定引物2条引物之间形成5’端互补序列的势能远远大于自身回文结构，所以就算3’端和5’端有互补的序列也会优先形成5’端二聚体结构。
2、利用本发明的引物对进行PCR扩增能够增加PCR扩增的特异性，有效降低非特异性产物的产生；其从PCR的第二个循环开始，引物5’端碱基(随机序列)和新生成的模板的5’端的碱基反向互补结合，引物的3’端特异性序列和新生成的模板3’端的碱基反向互补结合，也即引物和模板结合识别位点为2个(如附图2)，由此，显著增加了引物和模板的结合能力，以及扩增的特异性。而引物和模板的结合率提高，扩增效率也得到了有效的提高。
3、利用本发明的PCR引物对进行PCR扩增，能够有效降低测序文库(尤其是二代测序文库)扩增中不同模板的GC偏向性，因为PCR扩增只有在模板变性后引物结合上去才能发生有效的扩增。GC偏好性的产生，是因为在一些高GC区域，PCR过程中模板复性很快，模板在引物还没有结合上去就复性了，导致这些区域不能进行有效扩增。而本发明的锁定引物和模板有2个结合位点，这样能够大大提高和模板的结合能力，进而能够有效结合那些高GC的模板，从而降低GC偏向性。
4、本发明的定量PCR扩增方法得到的产物为带缺口的类环状物(即该类环状物的5’端和3’端未连接)，对于一些产物需要进行环化操作的实验来讲只需加入连接酶即可实现环化，不需要复杂的变性、淬火过程，能够有效简化实验流程。
5、本发明的定量PCR扩增方法，依赖于用于定量PCR扩增的组合物中的特殊设计的PCR引物对。相对于常规的qPCR，利用本发明的组合物进行qPCR时，特异性更强、扩增效率更高，用于DNA或RNA的定量分析、基因表达差异分析时，结果更加精确、可靠；并且本发明的组合物用于qPCR多重扩增时具有非常好的均一性，对多位点qPCR检测具有一定的优势。
6、本发明的用于定量PCR扩增的组合物、定量PCR扩增试剂盒及定量PCR扩增方法，可广泛用于DNA或RNA的定量分析、基因表达差异分析、基因分型、病原体检测等领域。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1显示了根据本发明一个实施例，本发明的PCR引物对(即锁定引物)的结构示意图；
图2显示了根据本发明的实施例，本发明环状扩增中引物与新生成链的结合示意图；
图3显示了根据本发明的实施例，利用本发明的组合物进行定量PCR扩增的流程示意图；
图4显示了实施例1中，常规SRY引物PCR扩增产物的琼脂糖凝胶电泳检测结果；
图5显示了实施例1中，PP引物扩增荧光检测信号；
图6显示了实施例1中，常规引物扩增荧光检测信号；
图7显示了实施例1中，PP引物CT值标准曲线图；
图8显示了实施例1中，常规引物CT值标准曲线图；
图9显示了实施例2中，各样本的qPCR荧光信号检测结果。
发明详细描述
下面详细描述本发明的实施例，所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。
需要说明的是，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。进一步地，在本发明的描述中，除非另有说明，“多个”的含义是两个或两个以上。
用于定量PCR扩增的组合物
在本发明的第一方面，本发明提供了一种用于定量PCR扩增的组合物。根据本发明的实施例，该用于定量PCR扩增的组合物包括：一对PCR引物对和一条特异性探针，所述PCR引物对包括第一引物和第二引物，其中，所述第一引物包含第一特异性序列和第一随机序列，所述第一特异性序列位于所述第一引物的3’端，所述第一随机序列位于所述第一引物的5’端，所述第二引物包含第二特异性序列和第二随机序列，所述第二特异性序列位于所述第二引物的3’端，所述第二随机序列位于所述第二引物的5’端，并且，所述第一特异性序列和所述第二特异性序列分别为针对靶序列的上游引物和下游引物，所述第一随机序列和所述第二随机序列反向互补，所述特异性探针的5’末端连接有荧光基团，3’末端连接有淬灭基团，且所述特异性探针的序列与所述靶序列的上游引物和下游引物之间的序列互补配对。发明人惊奇地发现，本发明的组合物中，该PCR引物对能有效降低PCR扩增过程中的GC偏向性，提高扩增特异性。具体地，用常规引物在二代测序文库PCR富集过程会带来一定的GC偏向性，而本发明的PCR引物对(有时也称为“锁定引物”)能有效降低文库PCR富集过程中的GC偏向性。进而，利用包含该PCR引物对及对应的特异性探针的组合物进行qPCR时，相对于常规的qPCR，特异性更强、扩增效率更高，用于DNA或RNA的定量分析、基因表达差异分析时，结果更加精确、可靠。并且本发明的组合物用于qPCR多重扩增时具有非常好的均一性，对多位点qPCR检测具有一定的优势。
其中，需要说明的是，本发明的“第一随机序列”和“第二随机序列”可以是随机序列，也可以是固定序列，只要保证两者反向互补即可。
根据本发明的实施例，所述第一特异性序列和所述第二特异性序列的TM值为55-65摄氏度，所述第一引物和所述第二引物的TM值为65-75摄氏度。由此，能够使该PCR先进行第一轮低退火温度(55-65摄氏度)的线状扩增，然后在后面的循环中采用高退火温度(65-72摄氏度)——即进行第二轮环状扩增。由于在环状扩增过程中，该轮退火温度高，特异性序列不能够单独结合到特异性位点(特异性序列的TM值只有55-65摄氏度)，只有当锁定引物的5’端结合到模板的5’端，同时该锁定引物的3’端结合到模板的特异性位点，该PCR才能够进行有效的扩增，也即该轮扩增实际为双结合位点的环状扩增。
本发明的PCR引物对适用于针对任何形式的待测DNA样品进行PCR扩增和文库构建。其中，需要说明的是，在本发明中所述的“待测DNA样品”和常规的理解有些不同，常规理解不包含处理过的DNA。但在本发明中，“待测DNA样品”可包括处理过的DNA以及未经处理的DNA(在构建测序文库时，一般会针对样本的基因组DNA进行打断和加测序接头处理，以得到携带相应平台测序接头的DNA片段，再经过后续扩增等步骤后，得到的产物即可用于测序，该携带相应平台测序接头的DNA片段即为“处理过的DNA片段”，相应地，为经过上述处理的，即为“未经处理的DNA”)。如果是针对未经处理的DNA，利用本发明的PCR引物对扩增，即是针对特异目标片段进行扩增；若是针对处理过的DNA，扩增的目标片段可以为整个基因组的DNA片段。
根据本发明的一些实施例，当待测DNA样品为处理过的、携带通用序列(例如测序接头)的DNA片段(这里的“通用序列”是用于与引物中特异序列配对的序列，包括测序平台接头序列，也即测序接头)时，相应地，所述第一特异性序列和所述第二特异性序列必须能够特异性识别携带通用序列的靶序列，换言之，实际上此时的靶序列实际为“通用序列+目标区域序列”；当待测DNA样品(也即PCR反应模板)为不携带通用序列的DNA片段时，相应地，所述第一特异性序列和所述第二特异性序列能够特异性识别该靶序列即可。并且，此时，如果需要构建测序文库，则可以在第一引物和第二引物的随机序列中或者特异性序列和随机序列之间设置测序接头序列(即通用序列)，以便使PCR扩增产物上连接测序接头，进而能够有效用于测序平台。
根据本发明的另一些实施例，所述第一引物和所述第二引物的至少之一进一步包含标签序列，由此，可以同时对多个样本进行PCR扩增，并基于所述标签序列对各个样本进行区分。其中，所述标签序列在第一引物和第二引物中的位置没有特别限制，只要能够使其发挥区分各样本的作用，且不影响PCR扩增进行即可。根据本发明的一些具体示例，所述标签序列可以位于特异性序列和随机序列之间，由此，可以在第一引物的第一特异性序列和第一随机序列之间设置一个标签序列，和/或，在第二引物的第二特异性序列和第二随机序列之间设置一个标签序列。根据本发明的另一个实施例，所述标签序列还可以被设置为包含于随机序列中，也即为随机序列的一部分。由此，同样能够使其发挥区分各样本的作用，且并不影响PCR扩增的进行。
根据本发明的实施例，所述第一随机序列和所述第二随机序列的长度为16-30bp，所述第一特异性序列和所述第二特异性序列的长度为16-30bp。
根据本发明的实施例，所述第一引物和所述第二引物的5’末端和3’末端的第1-5个碱基经过硫代修饰。由此，能够有效防止酶的外切。
根据本发明的一些实施例，硫代修饰的种类不受特别限制，只要能够防止第一引物和所述第二引物被酶外切(例如被具有5’-3’或3’-5’外切酶活性的降解)即可。根据本发明的一些具体示例，所述硫代修饰为选自硫代磷酸型修饰、甲基硫酸型修饰和肽核酸修饰的任意一种。
根据本发明的实施例，所述第一引物和所述第二引物的至少之一的5’端经过磷酸化修饰。由此，经过两轮扩增得到的带有缺口的类环状物(即类环状物的5’端和3’端未连接)，通过加入连接酶即可进行连接反应而形成完整的环状DNA。也即，可以基于qPCR的产物制备环状DNA文库。
此外，需要说明的是，参照图1中本发明的PCR引物对的结构示意图可知，本发明的PCR引物对的设计策略是：在一对常规引物(包括正向引物、反向引物)的5’端加上一段互补的序列，这段互补序列可以是一段随机序列也可以是一段固定序列，由此，即将PCR的一对引物设计成了5’端反向互补，3’端突出的“锁定引物”(PadlockPrimer，PP)，锁定引物的引物对之间形成了一个稳定的引物二聚体结构。整个引物对每条链的长度为32-60bp，TM值较高(第一引物和第二引物的TM值一般在65-75摄氏度)，其中锁定引物的5’端互补序列(即第一随机序列和第二随机序列)长度为16-30bp，其序列可以是随机的也可以是固定的；3’端(即第一特异性序列和第二特异性序列)长度为16-30bp，其和模板的靶序列互补，TM值较低(一般为55-65摄氏度)。
另外，针对本发明的PCR引物对的应用问题，本发明的PCR引物对需要通过2个不同的扩增部分(即两轮扩增)来实现PCR扩增(参照图3)：在第一个扩增部分，退火温度为55-65℃，循环数为1；在第二个扩增部分，退火温度为65-72℃，循环数为40-50。在第一个扩增部分中，PCR引物对只能通过3’端的特异性序列和模板结合，因此该循环的退火温度低；在第二个扩增部分中，PCR引物对先通过5’端互补的序列(即第一随机序列和第二随机序列)和新生成的模板(即第一轮扩增的产物)结合，再通过3’端的特异性序列(即第一特异性序列和第二特异性序列)和新生成的模板结合，也即引物和模板结合识别位点为2个，而2个锚定位点的结合大大提高了引物的退火温度，因此退火温度较高。
此外，需要说明的是，在第二个扩增部分中，引物5’端和3’端只有同时和新生成的模板结合时才能够进行有效的环状扩增，这样，2个识别位点大大提高了该种PCR扩增的特异性，2个结合位点大大提高了引物和模板的结合能力，提高了该PCR的扩增效率。由此，相对于传统PCR引物，采用本发明的PCR引物对进行PCR扩增，能够显著增加PCR扩增的特异性，有效降低非特异性产物的产生，并降低扩增过程中的GC偏向性。由此，在测序中尤其是二代测序文库中应用该种引物，可以有效降低文库富集扩增中全基因组范围内的GC偏向性。
并且，根据本发明的实施例，采用本发明的PCR引物对的PCR扩增可以应用于产物环化，得到的产物可以直接进行连接，得到环状DNA。具体地，不需要进行额外的变性、淬火等步骤，产物只要加入连接反应体系即可成环，由此，能够大大简化成环的流程，进而简化环状DNA文库的制备流程。
根据本发明的实施例，所述特异性探针的序列长度为18-30bp，TM值为70-80摄氏度。
应用
进而，在本发明的第二方面，本发明提供了一种定量PCR扩增试剂盒。根据本发明的实施例，该试剂盒包含前面所述的用于定量PCR扩增的组合物。根据本发明的实施例，相对于常规的qPCR，利用包含本发明的组合物进行qPCR时，特异性更强、扩增效率更高，用于DNA或RNA的定量分析、基因表达差异分析时，结果更加精确、可靠。并且本发明的组合物用于qPCR多重扩增时具有非常好的均一性，对多位点qPCR检测具有一定的优势。
进一步，本发明提出了组合物及包含它的试剂盒的应用。
在本发明的第三方面，本发明提供了一种定量PCR扩增方法。根据本发明的实施例，该方法利用前面所述的用于定量PCR扩增的组合物或者定量PCR扩增试剂盒进行所述定量PCR扩增。由此，利用该方法能够有效实现模板的定量PCR扩增。并且该方法能够增加定量PCR扩增的特异性，有效降低非特异性产物的产生，并提高扩增效率，进而能够提高定量结果的准确度。
根据本发明的实施例，所述方法包括如下的两轮扩增：于55-65摄氏度的退火温度下，在特异性探针存在时，使PCR引物对和模板进行第一轮线状扩增；以及于65-72摄氏度的退火温度下，对第一轮线状扩增的产物进行第二轮环状扩增。这样，从PCR的第二个循环(也即第二轮环状扩增)开始，第一引物和第二引物的5’端碱基和新生成的模板5’端的碱基反向互补结合，第一引物和第二引物的3’端特异性碱基和新生成的模板3’端的碱基反向互补结合，也即引物和模板结合识别位点为2个(如附图2)，从而能够增加PCR扩增的特异性，并有效降低非特异性产物的产生。
根据本发明的实施例，所述方法的扩增反应程序如下：
Figure PCTCN2017089198-appb-000001
由此，PCR扩增过程中的GC偏向性低、扩增特异性高，扩增效果好。
在本发明的第四方面，本发明提供了一种对待测DNA样品进行定量分析的方法。根据本发明的实施例，该方法包括：根据前面所述的定量PCR扩增方法，将待测DNA样品进行荧光定量PCR扩增，并基于采集的荧光信号实现定量分析。由此，PCR扩增的特异性好，扩增效率高，定量分析结果准确可靠。
在本发明的第五方面，本发明提供了一种对多个待测DNA样品进行特定基因的基因表达差异分析的方法。根据本发明的实施例，所述多个待测DNA样品均包含所述特定基因的cDNA序列，所述方法包括：根据前面所述的定量PCR扩增方法，分别将所述多个待测DNA样品进行荧光定量PCR扩增，并基于采集的荧光信号实现定量分析；以及比较多个待测DNA样品的定量分析结果，以便确定多个待测DNA样品的特定基因的基因表达差异。由此，基因表达差异分析的结果准确可靠。
此外，本发明的定量PCR扩增方法得到的产物为带缺口的类环状物(即该类环状物的5’端和3’端未连接)，对于一些产物需要进行环化操作的实验来讲只需加入连接酶即可实现环化，不需要复杂的变性、淬火过程，能够有效简化实验流程。
进而，在本发明的另一方面，本发明还提供了一种制备环状DNA文库的方法。根据本发明的实施例，该方法包括以下步骤：
(1)根据前面所述的定量PCR扩增方法，将待测DNA样品进行定量PCR扩增，以便获得包含类环状物的扩增产物，其中，所述类环状物的5’端和3’端未连接，所述第一引物和所述第二引物的至少之一的5’端经过磷酸化修饰，且所述第一引物和所述第二引物的5’末端和3’末端的第1-5个碱基经过硫代修饰；以及
(2)利用连接酶将所述扩增产物进行连接反应，以便使所述类环状物的5’端和3’端连接，形成环状DNA，所有环状DNA构成所述环状DNA文库，
其中，当所述第一引物和所述第二引物的其中之一的5’端经过磷酸化修饰时，所述环状DNA文库为单链环状DNA文库，当所述第一引物和所述第二引物的5’端均经过磷酸化修饰时，所述环状DNA文库为双链环状DNA文库。
根据本发明的实施例，利用该方法能够有效制备单链或双链的环状DNA文库，并且，获得的单链或双链的环状DNA文库，文库质量好，用于DNA保存或文库测序时效果好。
根据本发明的实施例，进一步包括：(3)去除线性DNA。由此，获得的文库质量好。
根据本发明的一些具体示例，利用线性消化反应去除线性DNA。
根据本发明的实施例，在步骤(1)中，所述待测DNA样品两端添加有通用序列。如前所述，本文所使用的表达方式“通用序列”是指用于与引物中特异序列配对的序列，包括测序平台接头序列，也即测序接头。由此，当待测DNA样品两端添加有通用序列例如测序接头时，获得的文库能够直接用于对应的测序平台进行上机测序。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分 子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品，例如可以采购自Illumina公司。
实施例1 PP-qPCR扩增效率测试
1.qPCR模板制备
取男性基因组DNA 10ng(基因组来自美国ATCC公司，货号Human genomic DNA(PC-3)(
Figure PCTCN2017089198-appb-000002
CRL-1435DTM))用常规SRY引物进行PCR扩增：
扩增体系如下：
Figure PCTCN2017089198-appb-000003
引物SRY F、SRY R序列见下表4。
反应条件如下：
Figure PCTCN2017089198-appb-000004
2.产物质检
得到的PCR产物经过XP Beads纯化(美国贝克曼公司Agencourt AMPure XP，货号：A63880)，纯化后进行琼脂糖电泳(见图4)，质检合格的PCR产物，备用
如图4所示，条带NTC为阴性对照(水为模板)，条带1、2为男性DNA扩增得到的产物，产物大小79bp，检测合格。
3.qPCR扩增效率测试
取步骤2得到的产物在通风处分别稀释10倍、100倍、1000倍、10000倍和100000倍5个梯度，待用，得到DNA分别为原模板浓度的10％，1％，0.1％，0.01％，0.001％；
取上述得到的梯度模板分别用PP引物(引物PP-SRY F和PP-SRY R，引物序列见下表4，结构示意图如图1)和常规引物(引物SRY F和SRY R，序列见下表4)进行qPCR反应，每个梯度重复三个反应，两种引物及其探针序列如表4。
用takara公司的Premix Ex TaqTM(货号RR390A)试剂盒进行qPCR反应，反应体系如下：
锁定引物反应体系：
Figure PCTCN2017089198-appb-000005
常规引物反应体系：
Figure PCTCN2017089198-appb-000006
在ABI公司的Stepone上进行qPCR反应，反应条件如下：
锁定引物反应条件：
Figure PCTCN2017089198-appb-000007
Figure PCTCN2017089198-appb-000008
常规引物反应条件：
Figure PCTCN2017089198-appb-000009
其中，利用本发明的PP引物进行qPCR扩增的流程如图3所示。
两种引物的检测结果见图5-图6。其中，图5为PP引物扩增荧光检测信号，图6为常规引物扩增荧光检测信号。
表1PP引物CT值：
Figure PCTCN2017089198-appb-000010
根据表1的统计数值绘制标准曲线，结果见图7。即图7显示了PP引物标准曲线。如图7所示，PP引物扩增效率E＝10-1/K＝1.9876。
表2常规引物CT值：
Figure PCTCN2017089198-appb-000011
根据表2的统计数值绘制标准曲线，结果见图8。即图8显示了常规引物标准曲线。如图8所示，常规引物扩增效率E＝10-1/K＝1.9872。
结论：PP引物在相同浓度模板检测中，荧光检测CT值比常规引物提前2个，扩增效率略高于常规引物。
实施例2 PP-qPCR对低孕周孕妇血浆游离DNA中胎儿特异性Y染色体的鉴定
1.引物设计
在Y染色体上的特异性基因SRY和DSY14上设计两对锁定引物(即PP引物，引物结构示意图如图1)及对应的探针，在常染色体上GADPH基因上设计一对锁定引物和探针作为实验和定量对照，各锁定引物及对应探针的序列见表4。其中，Y染色体上特异的探针标记的荧光基团和淬灭基团分别为FAM和BHQ1，GADPH基因上的探针标记的荧光基团和淬灭基团分别为HEX和BHQ1，用这3对引物进行一个多重qPCR对血浆中胎儿特异性Y染色体进行鉴定。
2.qPCR模板制备
挑选孕周分别为6/8/10周的孕妇血浆600ul，不同孕周男女胎各一例，用Qiagen血浆游离DNA抽提试剂盒抽提(QIAamp Circulating Nucleic Acid Kit，货号Cat No./ID：55114)，得到的血浆游离DNA溶于25ul AE中，用PP引物进行qPCR反应，其中本试验以水为对照。
其中，利用本发明的PP引物进行qPCR扩增的流程如图3所示。
具体地，用Takara公司的Premix Ex TaqTM(RR390A)试剂盒进行qPCR反应，反应体系如下：
Figure PCTCN2017089198-appb-000012
备注：正向引物混合物(10μM)：PP-SRY F、PP-DSY14F和PP-GADPH F混合物，各引物浓度分别为10μM；
反向引物混合物(10μM))：PP-SRY R、PP-DSY14R和PP-GADPH R混合物，各引物浓度分别为10μM；
探针混合物：SRY-Probe、DSY14-Probe和GADPH-Probe混合物，各探针浓度分别为μM)
在ABI公司的Stepone上进行qPCR反应，反应条件如下：
Figure PCTCN2017089198-appb-000013
荧光信号检测结果见图9。
qPCR检测得到的CT值如下表3：
表3
Figure PCTCN2017089198-appb-000014
结论：6个样本中均可以检测到HEX荧光，且CT值相差不大，说明cfDNA均抽提成功，且总cfDNA的量相差不大；水中没有检测到荧光；其中，三个男胎血浆样本均可检测到FAM荧光，且随着孕周的增加CT值有下降，三个女胎血浆样本均未检测到FAM荧光；该方法可以通过低至6周的孕妇血浆游离DNA正确检测胎儿性别。
表4引物序列
引物标号 序列(5’-3’，SEQ ID NO：)
PP-SRY F C-s-CCGCCCGTAGCCCGGGGCCAATGTTACCCGATTGTC-s-C(1)
PP-SRY R C-s-CCCGGGCTACGGGCGGGATCAACGCAGCCAGCTC-s-A(2)
SRY F CCAATGTTACCCGATTGTCC(3)
SRY R ATCAACGCAGCCAGCTCA(4)
SRY-Probe FAM-GGCTGTAGCGGTCCCGTTGCTGC-BHQ1(5)
PP-DSY14F G-s-CCGCGCGTACGGCGGGGCTGAGGGCTCGCTGACCTA-s-C(6)
PP-DSY14R C-s-CCCGCCGTACGCGCGGCCACCCACGCCACAGAAAC-s-C(7)
DSY14-Probe FAM-GGTGCCAGAGAGGCTGCGG-BHQ1(8)
PP-GADPH F G-s-CCCGCGGTTGCCGGCCGTCGTCGCCCACATAGGAAT-s-C(9)
PP-GADPH R C-s-GGCCGGCAACCGCGGGCGTGCTCAGGGCTTCTTGTC-s-C(10)
GADPH-Probe HEX-TGCCCACCATCACGCCCTG-BHQ1(11)
备注：F：正向引物；R：反向引物
上表中各引物序列以下划线标注的部分为互补序列。
工业实用性
本发明的用于定量PCR扩增的组合物，能够有效地用于待测DNA样品的定量PCR扩增，并且相对于常规qPCR，特异性更强、扩增效率更高，用于DNA或RNA的定量分析、基因表达差异分析时，结果更加精确、可靠。并且本发明的组合物用于qPCR多重扩增时具有非常好的均一性，对多位点qPCR检测具有一定的优势。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示意性实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。

Claims (13)

  1. 一种用于定量PCR扩增的组合物，其特征在于，包括：一对PCR引物对和一条特异性探针，
    所述PCR引物对包括第一引物和第二引物，
    其中，
    所述第一引物包含第一特异性序列和第一随机序列，所述第一特异性序列位于所述第一引物的3’端，所述第一随机序列位于所述第一引物的5’端，
    所述第二引物包含第二特异性序列和第二随机序列，所述第二特异性序列位于所述第二引物的3’端，所述第二随机序列位于所述第二引物的5’端，
    并且，
    所述第一特异性序列和所述第二特异性序列分别为针对靶序列的上游引物和下游引物，所述第一随机序列和所述第二随机序列反向互补，
    所述特异性探针的5’末端连接有荧光基团，3’末端连接有淬灭基团，且所述特异性探针的序列与所述靶序列的上游引物和下游引物之间的序列互补配对。
  2. 根据权利要求1所述的组合物，其特征在于，所述第一特异性序列和所述第二特异性序列的TM值为55-65摄氏度，所述第一引物和所述第二引物的TM值为65-75摄氏度。
  3. 根据权利要求1所述的组合物，其特征在于，所述第一随机序列和所述第二随机序列的长度为16-30bp，所述第一特异性序列和所述第二特异性序列的长度为16-30bp。
  4. 根据权利要求1所述的组合物，其特征在于，所述第一引物和所述第二引物的5’末端和3’末端的第1-5个碱基经过硫代修饰。
  5. 根据权利要求4所述的组合物，其特征在于，所述硫代修饰为选自硫代磷酸型修饰、甲基硫酸型修饰和肽核酸修饰的任意一种。
  6. 根据权利要求1所述的组合物，其特征在于，所述第一引物和所述第二引物的至少之一的5’端经过磷酸化修饰。
  7. 根据权利要求1所述的组合物，其特征在于，所述特异性探针的序列长度为18-30bp，TM值为70-80摄氏度。
  8. 一种定量PCR扩增试剂盒，其特征在于，包含权利要求1-7任一项所述的用于定量PCR扩增的组合物。
  9. 一种定量PCR扩增方法，其特征在于，利用权利要求1-7任一项所述的用于定量PCR扩增的组合物或者权利要求8所述的定量PCR扩增试剂盒进行所述定量PCR扩增。
  10. 根据权利要求9所述的方法，其特征在于，所述方法包括如下的两轮扩增：
    于55-65摄氏度的退火温度下，在特异性探针存在时，使PCR引物对和模板进行第一轮线状扩增；以及
    于65-72摄氏度的退火温度下，对第一轮线状扩增的产物进行第二轮环状扩增。
  11. 根据权利要求10所述的方法，其特征在于，所述方法的扩增反应程序如下：
    Figure PCTCN2017089198-appb-100001
  12. 一种对待测DNA样品进行定量分析的方法，其特征在于，包括：
    根据权利要求9-11任一项所述的方法，将待测DNA样品进行荧光定量PCR扩增，并基于采集的荧光信号实现定量分析。
  13. 一种对多个待测DNA样品进行特定基因的基因表达差异分析的方法，其特征在于，所述多个待测DNA样品均包含所述特定基因的cDNA序列，所述方法包括：
    根据权利要求9-11任一项所述的方法，分别将所述多个待测DNA样品进行荧光定量PCR扩增，并基于采集的荧光信号实现定量分析；以及
    比较多个待测DNA样品的定量分析结果，以便确定多个待测DNA样品的特定基因的基因表达差异。
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