WO2018232595A1 - Pcr引物对及其应用 - Google Patents

Pcr引物对及其应用 Download PDF

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WO2018232595A1
WO2018232595A1 PCT/CN2017/089196 CN2017089196W WO2018232595A1 WO 2018232595 A1 WO2018232595 A1 WO 2018232595A1 CN 2017089196 W CN2017089196 W CN 2017089196W WO 2018232595 A1 WO2018232595 A1 WO 2018232595A1
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primer
sequence
pcr
pcr amplification
amplification
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PCT/CN2017/089196
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English (en)
French (fr)
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WO2018232595A8 (zh
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杨林
黄国栋
高雅
张艳艳
张海萍
陈芳
蒋浩君
张韶红
王雨倩
谢一帆
蒋慧
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深圳华大智造科技有限公司
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Priority to EP17914886.1A priority Critical patent/EP3643787A4/en
Priority to PCT/CN2017/089196 priority patent/WO2018232595A1/zh
Priority to US16/624,779 priority patent/US11739319B2/en
Priority to CN201780090470.3A priority patent/CN110603334B/zh
Publication of WO2018232595A1 publication Critical patent/WO2018232595A1/zh
Publication of WO2018232595A8 publication Critical patent/WO2018232595A8/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
    • 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
    • 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
    • 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
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the present invention relates to the field of biotechnology, and in particular to PCR amplification, and more particularly to PCR primer pairs and their applications.
  • Primers are one of the key factors in all PCR method technologies. Primer designs are currently available through computers and networks. The advantages of the online primer design are: 1) no need to be proficient in software operation; 2) more variables can be analyzed simultaneously. However, in practice, good primers designed by conventional primer design software do not necessarily produce good results, especially those with high GC regions and similar sequences in the template, which ultimately lead to amplification of the primers. The specificity is not strong, and the PCR amplification efficiency is not high, and it is often difficult to obtain satisfactory results by optimizing primers in these regions.
  • an object of the present invention is to propose a PCR primer design strategy and a corresponding PCR primer pair which can effectively reduce GC bias during PCR amplification and improve amplification specificity.
  • 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.
  • a sequence not included in the target sequence is set between the conventional primer and the complementary sequence as a predetermined restriction site, and the primer can be efficiently removed by performing restriction enzyme digestion on the amplified product after PCR. And complementary sequences facilitate subsequent library construction and purification.
  • the invention provides a PCR primer pair.
  • the PCR primer pair comprises: 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 in the a 3' end of the first primer, the first random sequence is located at the 5' end of the first primer, and the second primer comprises a second specific sequence and a second random sequence, the second specific sequence is located a 3' end of the second primer, the second random sequence is located at the 5' end of the second primer, and the first specific sequence and the second specific sequence are respectively directed to a target sequence
  • An upstream primer and a downstream primer wherein the first random sequence and the second random sequence are inversely complementary, and a predetermined restriction site is ligated between the first specific sequence and the first random sequence, A predetermined restriction site is ligated between the bispecific sequence and the second random sequence, and the target sequence of the PCR primer pair does not comprise the predetermined restriction site.
  • the PCR primer pair of the present invention can effectively reduce the GC bias during PCR amplification and increase the 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 the PCR primer pair of the present invention sets a sequence which is not included in the target sequence between the conventional primer and the complementary sequence as a predetermined restriction site, and can be efficiently cut by a predetermined restriction site of the amplification product after PCR. Removal of primers and complementary sequences facilitates subsequent library construction and purification.
  • the PCR primer pair of the present invention is particularly suitable for multiplex PCR amplification, that is, the PCR primer pair of the present invention designed for a plurality of target sequences, which can be effectively mixed, that is, multiplex PCR.
  • the invention provides a PCR amplification kit.
  • the kit comprises the PCR primer pair described above.
  • the PCR primer pair of the present invention is used for PCR amplification using the kit with respect to the conventional primer, and the GC biasing property during the amplification process is low, the amplification specificity is high, and the amplification effect is outstanding.
  • the PCR primer pair of the present invention is particularly suitable as a primer for multiplex PCR amplification.
  • the invention provides a method of PCR amplification.
  • the method performs the PCR amplification using a PCR primer pair or a PCR amplification kit as described above.
  • PCR amplification of the template can be effectively achieved by this method.
  • the method can increase the specificity of PCR amplification, effectively reduce the production of non-specific products, and improve the amplification efficiency.
  • the invention provides a method of preparing a DNA library. According to an embodiment of the invention, the method comprises the steps of:
  • a first PCR amplification product comprising a loop-like substance, wherein the first specific sequence and a predetermined restriction site is ligated between the first random sequence, a predetermined restriction site is ligated between the second specific sequence and the second random sequence, and the target sequence of the PCR primer pair is not Including the predetermined restriction site, the 5' end and the 3' end of the loop-like substance are not linked, and the first primer of the PCR primer pair and the first primer of the second primer and the first primer of the 3' end - 5 bases are thiolated;
  • the first PCR amplification product is subjected to enzymatic cleavage using an enzyme corresponding to the predetermined restriction site, and library preparation is performed based on the digested product to obtain a DNA library of interest.
  • a linear DNA library or a circular DNA library can be efficiently prepared by the method, and the obtained DNA library has a good library quality and is effective for DNA preservation or library sequencing.
  • the PCR primer pair of the invention and its use 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 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. Moreover, 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.
  • the PCR amplification method 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 newly generated The base of the 5' end of the 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 (eg attached) Figure 3), thus, significantly increases the binding ability of the primer and template, as well as the specificity of amplification. 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 template have two binding sites, which can greatly improve the binding ability of the template, and thus can effectively combine those high GC templates, thereby reducing the GC bias.
  • the PCR amplification pair of the present invention which sets a sequence not included in the target sequence between a conventional primer (ie, a specific sequence) and a complementary sequence (ie, a random sequence) as a predetermined restriction site, so as to be able to pass after PCR.
  • Enzyme digestion of the amplified product at a predetermined restriction site can effectively remove the primer and the complementary sequence, thereby facilitating subsequent library construction and purification.
  • the predetermined restriction site is the base U of the cleavage site of the USER enzyme, whereby the enzyme digestion can be conveniently and efficiently performed.
  • 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 view showing the structure of a PCR primer pair (ie, a locked primer) of the present invention according to another embodiment of the present invention
  • Figure 3 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. 4 is a schematic flow diagram showing the preparation of a linear library using the PCR primer pair of the present invention, in accordance with an embodiment of the present invention
  • Figure 5 shows the fragment analysis and qubit quantitative detection results of the target library of each sample obtained in Example 1;
  • Figure 6 is a graph showing the sequencing result of the sequencing of different amplicon of the objective library of each sample obtained in Example 1, (i.e., the result of homogeneity detection);
  • Fig. 7 shows the comparison result (i.e., the stability detection result) of the same position depth in the two experiments in Example 1.
  • 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 PCR primer pair.
  • the PCR primer pair comprises: a first primer and a second primer, wherein the first primer comprises a first specific sequence and a first random a 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, and the second primer comprises a second specific sequence and a a second random sequence, the second specific sequence is located at the 3' end of the second primer, the second random sequence is located at the 5' end of the second primer, and the first specific sequence and The second specific sequence is an upstream primer and a downstream primer respectively for the target sequence, the first random sequence and the second random sequence are inversely complementary, the first specific sequence and the first random sequence a predetermined restriction site is ligated, a predetermined restriction site is ligated between the second specific sequence and the second random sequence, and the target sequence of the PCR primer pair does not comprise the predetermined cleavage site point.
  • the PCR primer pair of the present invention can effectively reduce the GC bias during PCR amplification and increase the 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 the PCR primer pair of the present invention sets a sequence which is not included in the target sequence between the conventional primer and the complementary sequence as a predetermined restriction site, and can be efficiently cut by a predetermined restriction site of the amplification product after PCR. Removal of primers and complementary sequences facilitates subsequent library construction and purification.
  • the 5' end of the digested product is phosphorylated, and the 3' end is a prominent terminal A, which does not require additional end repair and can be directly used for linker ligation.
  • the PCR primer pair of the present invention is particularly suitable for multiplex PCR amplification, that is, the PCR primer pair of the present invention designed for a plurality of target sequences, which can be effectively mixed, that is, multiplex PCR.
  • the PCR primers of the present invention have high specificity to themselves; 2.
  • the primers of different amplification regions have different and unique complementary random sequences, and the primers of the same amplified region are uniquely complementary.
  • the sequences are closely linked, avoiding mutual interference between primers in different amplification regions, and is advantageous for inhibiting the production of non-specific products.
  • 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, and 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.
  • the specific sequence cannot bind to the specific site alone (the specific sequence has a TM value of only 55-65), only when the 5' end of the locked primer binds 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 circular amplification of the double binding site .
  • 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 for 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 may 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 15-45 bp in length, and the first specific sequence and the second specific sequence are 15-30 bp in length.
  • the 5' end and the 1st base of the 3' end of the first primer and the second primer are modified to effectively prevent cleavage of the enzyme.
  • the first primer and the second primer and the 1-5th base of the 3' end are thio-modified.
  • the type of thio modification is not particularly limited as long as the first primer and the second primer are prevented from being exo-enzymatically cleaved (for example, by having 5-3' or 3-5' exonuclease The active enzyme is degraded).
  • 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 predetermined cleavage site is the cleavage site base U of the USER enzyme. Therefore, since the target sequence does not include the base U of the cleavage site of the USER enzyme, it is possible to efficiently cleave the amplified product after PCR, and remove the primer and the complementary sequence, which is advantageous for subsequent library construction and purification.
  • 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. Locking Primer" (PadlockPrimer, PP), lock A stable primer-dimer structure is formed between the primer pairs of the primers.
  • the length of the entire primer pair is 30-70 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 15-45 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 15-30 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 5-35. 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.
  • the invention also provides a PCR amplification kit.
  • the kit comprises the PCR primer pair described above.
  • the PCR primer pair of the present invention is used for PCR amplification using the kit with respect to the conventional primer, and the GC biasing property during the amplification process is low, the amplification specificity is high, and the amplification effect is outstanding.
  • the PCR primer pair of the present invention is particularly suitable as a primer for multiplex PCR amplification.
  • the present invention proposes the use of a PCR primer pair and a kit comprising the same.
  • the invention provides a method of PCR amplification.
  • the method performs the PCR amplification using a PCR primer pair or a PCR amplification kit as described above.
  • PCR amplification of the template can be effectively achieved by this method.
  • the method can increase the specificity of PCR amplification, effectively reduce the production of non-specific products, and improve the amplification efficiency.
  • the method comprises two rounds of amplification: the PCR primer pair and the template are subjected to a first round of linear amplification at an annealing temperature of 55-65 degrees Celsius; and at 65-72 At the annealing temperature of Celsius, a second round of circular amplification was performed on the first linearly amplified product.
  • 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 inversely complementary, that is, the primer and template binding recognition sites are 2 (as shown in FIG. 3), 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 of preparing a circular DNA library.
  • the invention provides a method of preparing a DNA library. According to an embodiment of the invention, the method comprises the steps of:
  • a first PCR amplification product comprising a loop-like substance, wherein the first specific sequence and a predetermined restriction site is ligated between the first random sequence, a predetermined restriction site is ligated between the second specific sequence and the second random sequence, and the target sequence of the PCR primer pair is not Including the predetermined restriction site, the 5' end and the 3' end of the loop-like substance are not linked, and the first primer of the PCR primer pair and the first primer of the second primer and the first primer of the 3' end - 5 bases are thiolated;
  • the first PCR amplification product is subjected to enzymatic cleavage using an enzyme corresponding to the predetermined restriction site, and library preparation is performed based on the digested product to obtain a DNA library of interest.
  • a linear DNA library or a circular DNA library can be efficiently prepared by the method, and the obtained DNA library has a good library quality and is effective for DNA preservation or library sequencing.
  • the predetermined cleavage site is the cleavage site base U of the USER enzyme. Therefore, since the target sequence does not include the base U of the cleavage site of the USER enzyme, it is possible to efficiently cleave the amplified product after PCR, and remove the primer and the complementary sequence, which is advantageous for subsequent library construction and purification.
  • the step (2) is carried out according to the following method: the first PCR amplification product containing the loop-like substance is subjected to the enzyme digestion and the terminal repair plus A to obtain the terminal-containing base A. Digesting the product; ligating the digested product comprising the base A at the end to obtain a ligation product, the ligation product is a linear DNA; performing second PCR amplification of the ligation product to obtain a second PCR amplification product, the second PCR amplification product
  • the composition constitutes a linear DNA library, wherein the second PCR amplification product is performed using sequencing primers that are complementary to the linker sequence.
  • the digestion and end repair plus A are performed using the USER enzyme, the T4 polynuclease, the T4 DNA polymerase, and the Klenow fragment.
  • the enzymatic cleavage effect is good, and the obtained library is high in quality.
  • the step (2) is carried out according to the following method: the first PCR amplification product containing the loop-like substance is subjected to the digestion to obtain a cleavage product comprising a cyclone; The cleavage product comprising the cyclase is subjected to a ligation reaction using a ligase to join the 5' end and the 3' end of the cyclized ring into a loop; the linear DNA in the ligation product is removed to obtain a loop a mixture of DNA; and subjecting the circular DNA mixture to a third PCR amplification to obtain a third PCR amplification product, the third PCR amplification product constituting a circular DNA library, wherein the third PCR amplification
  • the 3' end sequence of the increased forward primer is inversely complementary to the first random sequence
  • the 3' end sequence of the third PCR amplified reverse primer is inversely complementary to the second random sequence.
  • linear DNA is removed using a linear digestion reaction.
  • the step of purifying the circular DNA mixture is further included prior to performing the third PCR amplification.
  • the obtained library is of good quality.
  • Example 1 Using a locked primer method to detect whether a fetus has achondroplasia disease (ACH)
  • Primer design for locking primers ie, PCR primer pairs of the present invention, hereinafter referred to as "PP primers"
  • PP locked primer
  • F forward primer
  • R reverse primer
  • predetermined restriction site is set to the base U of the enzyme cleavage site of USER
  • for each primer, 5'-3', before 18 bp is a random sequence followed by a 1 bp base U and finally a specific sequence.
  • the positive and negative samples were taken for 200 ul.
  • the plasma DNA of pregnant women was extracted with magen beads, and the obtained product was dissolved in 17 ul of TE for use.
  • the library of interest was prepared by referring to the following procedure (schematic diagram of the preparation of the library is shown in Fig. 4).
  • the forward and reverse primer pools are obtained by mixing all the corresponding primers described in Table 2.
  • the amplification conditions are as follows:
  • step 1 98 ° C 2 min Step 2 98°C10s Step 3 58 ° C 2 min Step 4 72°C30s Step 5 98°C10s Step 6 68°C1min Step 7 Repeat steps 5-6, 25 cycles Step 8 72 ° C 5 min
  • the amplified products of each sample were separately purified with 1.8 volumes of anxygen beads and finally dissolved in 37 ul of TE for use.
  • reaction procedure of USER digestion and end repair plus "A” was: 20 ° C for 30 min; 65 ° C, 30 min.
  • the reaction procedure for the joint connection was: 25 ° C for 15 min; 4 ° C for 10 min.
  • the resulting product was then purified with 1.2 volumes of anxygen beads and finally dissolved in 21 TE for use.
  • PCR amplification was carried out according to the reaction system in the following table:
  • the sequence of the forward universal primer is (5'-3'):
  • the sequence of the reverse universal primer was (5'-3'): GAACGACATGGCTACGATCCGACTTGG (SEQ ID NO: 38).
  • the PCR amplification procedure is as follows:
  • the obtained product was purified by using 1.2 volumes of anxygen beads, and finally dissolved in 21TE to obtain a library of interest.
  • the obtained product was then subjected to fragment analysis and qubit quantification at 2100, and the results are shown in Fig. 5 and Table 3.
  • 1-5 is a positive sample, and 6-10 negative samples.
  • the library was qualified and subjected to sequencing on the machine.
  • the BGI seq500 sequencing platform was used, and the sequencing type was PE50BP.
  • the obtained sequencing results are analyzed by data, which specifically includes: filtering the sequencing reads of the lower machine, filtering out the reads having a base quality of less than 10, and ensuring that the data for analyzing the base frequency is high quality; using bwa (V0.
  • the comparison software compares the filtered clean reads back to the human genome reference sequence HG19 (GRCH37); calculates the coverage depth of the 4 base ATCG at the site to be detected based on the alignment result, and according to the base depth Obtain the most likely mutation site and its frequency, sequencing error and frequency, etc.; perform GC bias correction according to the GC distribution of the location of the site and the location of the primer and the possible sequencing error frequency; use the most accurate mutation detection tool GATK (V3.6) detects the variant sites obtained above and filters and annotates using the corresponding mutation database.
  • the inventors have found that using the PCR primers of the present invention for multiplex PCR amplification and library construction, it is possible to accurately detect mutations as low as 1%, specifically: in 10 samples, 5 positive samples were positive, detection The mutation frequencies were 5.3%, 7.2%, 3.5%, 5.6%, and 6.2%, respectively. Five negative samples were negative, and the detected mutation frequencies were 0.3%, 0.1%, 0.2%, 0.2%, and 0.1%, respectively. % (as in Table 5).
  • the method of the invention has good specificity, the alignment rate is between 90-94%, the capture efficiency is between 92-95%; the coverage is 100% (as shown in Table 4); the uniformity is good, and the average depth is 0.1X. More than 94% (see Table 4, Figure 6); good stability, small differences between different samples (see Figure 7).
  • the inventors also conducted a series of experiments, and found that the application range of the PCR primer pair of the present invention is not limited to cfDNA samples, and can be widely applied to samples such as genomic DNA, FFPE, urine DNA, fresh frozen, and not limited to construction.
  • the BGI-Seq library can also be used to construct other platform libraries such as illumina and proton.
  • the PCR primer pair of the invention can be effectively used for PCR amplification of a DNA sample to be tested, and can effectively reduce GC bias during PCR amplification, improve amplification specificity, and is particularly suitable for multiplex PCR amplification. Primer.

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Abstract

提供了PCR引物对及其应用。其中，该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，所述第一特异性序列和所述第一随机序列之间连接有预定酶切位点，所述第二特异性序列和所述第二随机序列之间连接有预定酶切位点。

Description

PCR引物对及其应用
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PCR扩增，更具体地，涉及PCR引物对及其应用。
背景技术
由Mullis在1983年建立的PCR方法已成为分子生物学及其相关领域的经典实验方法，其应用已趋于多元化，从基因扩增与基因检测，到基因克隆、基因改造、遗传分析，等等，甚至扩展到非生物学领域。随着近年的发展，该技术本身正不断进步，可靠性也不断提高。而同时，在聚合酶链式反应这一基本原理的基础上，还发展出了一系列新的概念和实验方法，它们在生命科学研究中有重要的应用价值。
引物是所有PCR方法技术的关键因素之一。目前，引物的设计可通过计算机和网络来实现。在线引物设计的优点在于：1)无须精通软件的操作；2)可同时分析更多的变量。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。
因而，目前的常规PCR引物设计方法仍有待改进。
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能有效降低PCR扩增过程中的GC偏向性，提高扩增特异性的PCR引物设计策略及相应的PCR引物对。
首先，需要说明的是，本发明是基于发明人的下列发现而完成的：
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。
并且，发明人还发现在常规引物和互补序列之间设置靶序列不包含的序列作为预定酶切位点，能够在PCR后，通过对扩增产物进行预定酶切位点的酶切有效去除引物和互补序列，有利于后续文库构建、纯化。
进而，在本发明的第一方面，本发明提供了一种PCR引物对。根据本发明的实施例，该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，所述第一特异性序列和所述第一随机序列之间连接有预定酶切位点，所述第二特异性序列和所述第二随机序列之间连接有预定酶切位点，且所述PCR引物对的靶序列不包含所述预定酶切位点。发明人惊奇地发现，本发明的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。并且，本发明的PCR引物对在常规引物和互补序列之间设置靶序列不包含的序列作为预定酶切位点，能够在PCR后，通过对扩增产物进行预定酶切位点的酶切有效去除引物和互补序列，有利于后续文库构建、纯化。此外，本发明的PCR引物对尤其适用于多重PCR扩增，也即针对多个靶序列设计获得的本发明的PCR引物对，能够有效地混合使用，即进行多重PCR。
在本发明的第二方面，本发明提供了一种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PCR引物对。根据本发明的实施例，相对于常规引物，利用该试剂盒采用本发明的PCR引物对进行PCR扩增，扩增过程中的GC偏向性低、扩增特异性高，扩增效果突出。并且，本发明的PCR引物对尤其适于作为多重PCR扩增的引物。
在本发明的第三方面，本发明提供了一种PCR扩增方法。根据本发明的实施例，该方法利用前面所述的PCR引物对或者PCR扩增试剂盒进行所述PCR扩增。由此，利用该方法能够有效实现模板的PCR扩增。并且该方法能够增加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。
在本发明的第四方面，本发明提供了一种制备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PCR扩增方法，将待测DNA样品进行第一PCR扩增，以便获得包含类环状物的第一PCR扩增产物，其中，所述第一特异性序列和所述第一随机序列之间连接有预定酶切位点，所述第二特异性序列和所述第二随机序列之间连接有预定酶切位点，且所述PCR引物对的靶序列不包含所述预定酶切位点，所述类环状物的5’端和3’端未连接，采用的PCR引物对的第一引物和第二引物的5’末端和3’末端的第1-5个碱基经过硫代修饰；以及
(2)利用所述预定酶切位点对应的酶，将所述第一PCR扩增产物进行酶切，并基于酶切产物进行文库制备，以便获得目的DNA文库。
根据本发明的实施例，利用该方法能够有效制备线性DNA文库或环状DNA文库，并且，获得的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，本发明的PCR引物对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。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、本发明的PCR扩增方法能够增加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3)，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PCR扩增对，在常规引物(即特异性序列)和互补序列(即随机序列)之间设置靶序列不包含的序列作为预定酶切位点，以便能够在PCR后，通过对扩增产物进行预定酶切位点的酶切即可有效去除引物和互补序列，从而有利于后续文库构建、纯化。其中，因靶序列不包含USER酶的酶切位点碱基U，设预定酶切位点为USER酶的酶切位点碱基U，由此，即能方便有效地实现酶切。
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显示了根据本发明另一个实施例，本发明的PCR引物对(即锁定引物)的结构示意图；
图3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4显示了根据本发明的实施例，利用本发明的PCR引物对制备线性文库的流程示意图；
图5显示了实施例1中，获得的各样本的目的文库的片段分析和qubit定量检测结果；
图6显示了实施例1中，获得的各样本的目的文库的不同扩增子的测序读段统计结果(即均一性检测结果)；
图7显示了实施例1中，两次实验同一个位置深度的比较结果(即稳定性检测结果)。
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。
PCR引物对
在本发明的第一方面，本发明提供了一种PCR引物对。根据本发明的实施例，该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，所述第一特异性序列和所述第一随机序列之间连接有预定酶切位点，所述第二特异性序列和所述第二随机序列之间连接有预定酶切位点，且所述PCR引物对的靶序列不包含所述预定酶切位点。发明人惊奇地发现，本发明的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。并且，本发明的PCR引物对在常规引物和互补序列之间设置靶序列不包含的序列作为预定酶切位点，能够在PCR后，通过对扩增产物进行预定酶切位点的酶切有效去除引物和互补序列，有利于后续文库构建、纯化。并且，酶切产物的5’端磷酸化，3’端是突出的末端A，不需要额外进行末端修复，直接可用于接头连接。
此外，本发明的PCR引物对尤其适用于多重PCR扩增，也即针对多个靶序列设计获得的本发明的PCR引物对，能够有效地混合使用，即进行多重PCR。这是因为：1、本发明的PCR引物对本身具有很高的特异性；2、不同扩增区域引物之间都有不同的、唯一的互补随机序列，同一扩增区域的引物通过唯一的互补序列紧密联系在一起，避免了不同扩增区域引物之间的互相干扰，有利于抑制非特异性产物的产生。
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)，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15-45bp，所述第一特异性序列和所述第二特异性序列的长度为15-30bp。
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个碱基经过修饰，用以有效防止酶的外切。根据本发明的一些具体示例，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。
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’外切酶活性的酶降解)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，所述预定酶切位点为USER酶的酶切位点碱基U。由此，因靶序列不包含USER酶的酶切位点碱基U，从而能够在PCR后，有效对扩增产物进行USER酶切，去除引物和互补序列，有利于后续文库构建、纯化。
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30-70bp，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15-45bp，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15-30bp，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5-35。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。
应用
进而，在本发明的第二方面，本发明还提供了一种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PCR引物对。根据本发明的实施例，相对于常规引物，利用该试剂盒采用本发明的PCR引物对进行PCR扩增，扩增过程中的GC偏向性低、扩增特异性高，扩增效果突出。并且，本发明的PCR引物对尤其适于作为多重PCR扩增的引物。
进一步，本发明提出了PCR引物对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PCR扩增方法。根据本发明的实施例，该方法利用前面所述的PCR引物对或者PCR扩增试剂盒进行所述PCR扩增。由此，利用该方法能够有效实现模板的PCR扩增。并且该方法能够增加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。
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，使所述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3)，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 PCTCN2017089196-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，本发明提供了一种制备环状DNA文库的方法。
在本发明的第四方面，本发明提供了一种制备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PCR扩增方法，将待测DNA样品进行第一PCR扩增，以便获得包含类环状物的第一PCR扩增产物，其中，所述第一特异性序列和所述第一随机序列之间连接有预定酶切位点，所述第二特异性序列和所述第二随机序列之间连接有预定酶切位点，且所述PCR引物对的靶序列不包含所述预定酶切位点，所述类环状物的5’端和3’端未连接，采用的PCR引物对的第一引物和第二引物的5’末端和3’末端的第1-5个碱基经过硫代修饰；以及
(2)利用所述预定酶切位点对应的酶，将所述第一PCR扩增产物进行酶切，并基于酶切产物进行文库制备，以便获得目的DNA文库。
根据本发明的实施例，利用该方法能够有效制备线性DNA文库或环状DNA文库，并且，获得的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，所述预定酶切位点为USER酶的酶切位点碱基U。由此，因靶序列不包含USER酶的酶切位点碱基U，从而能够在PCR后，有效对扩增产物进行USER酶切，去除引物和互补序列，有利于后续文库构建、纯化。
根据本发明的实施例，按照如下的方法进行步骤(2)：将所述包含类环状物的第一PCR扩增产物进行所述酶切以及末端修复加A，以便获得末端包含碱基A的酶切产物；将所述末端包含碱基A的酶切产物进行接头连接，以便获得连接产物，所述连接产物为线性DNA；将所述连接产物进行第二PCR扩增，以便获得第二PCR扩增产物，所述第二PCR扩增产 物构成线性DNA文库，其中所述第二PCR扩增产物采用测序引物进行，所述测序引物与所述接头序列互补。由此，能够有效获得线性DNA文库，且获得的文库质量非常好。
根据本发明的一些具体示例，利用USER酶、T4多聚核酸激酶、T4DNA聚合酶和Klenow片段进行所述酶切以及末端修复加A。由此，酶切效果好，获得的文库质量高。
根据本发明的实施例，按照如下的方法进行步骤(2)：将所述包含类环状物的第一PCR扩增产物进行所述酶切，以便获得包含类环状物的酶切产物；利用连接酶将所述包含类环状物的酶切产物进行连接反应，以便使所述类环状物的5’端和3’端连接成环；去除连接产物中的线性DNA，以便获得环状DNA混合物；以及将所述环状DNA混合物进行第三PCR扩增，以便获得第三PCR扩增产物，所述第三PCR扩增产物构成环状DNA文库，其中，所述第三PCR扩增的正向引物的3’端序列与所述第一随机序列反向互补，所述第三PCR扩增的反向引物的3’端序列与所述第二随机序列反向互补。由此，能够有效获得环状DNA文库，且获得的文库质量非常好。
根据本发明的实施例，利用线性消化反应去除线性DNA。
根据本发明的实施例，在进行所述第三PCR扩增之前，进一步包括对所述环状DNA混合物进行纯化的步骤。由此，获得的文库质量好。
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：用锁定引物方法检测胎儿是否患有软骨发育不全疾病(ACH)
一、锁定引物(即本发明的PCR引物对，下面简称为“PP引物”)引物设计
设计18对多重PCR引物(如表1，均为锁定引物)，包含软骨发育不全疾病所有突变位点，引物设计采用Primer3，3’端TM值在55-65摄氏度之间，且没经过任何优化，5’端互补序列随机生成，TM在65-75摄氏度之间，扩增子区域GC含量在26-75％之间(如表2，引物结构示意图如图1)。
表1：引物区间信息
Figure PCTCN2017089196-appb-000002
Figure PCTCN2017089196-appb-000003
注：PP：锁定引物；F：正向引物；R：反向引物。
表2：引物序列信息
Figure PCTCN2017089196-appb-000004
Figure PCTCN2017089196-appb-000005
注：PP：锁定引物；F：正向引物；R：反向引物；预定酶切位点设为USER酶的酶切位点碱基U；针对每条引物，由5’-3’，前18bp为随机序列，接着是1bp碱基U，最后是特异性序列。
二、实验步骤
本实验选取孕妇血浆10例进行实验，其中包括5例ACH阳性样本和5例ACH阴性样本。其中，实验设一次重复。
1.血浆游离DNA提取
分别取阳性样本和阴性样本血浆，用量200ul，用magen beads对孕妇血浆DNA进行提取，得到的产物溶于17ul TE中，备用。然后参照下述步骤制备目的文库(制备文库的流程示意图见图4)。
2.多重PCR扩增
针对提取得到的cfDNA的每一个，分别按照下表所示的扩增体系进行多重PCR：
Figure PCTCN2017089196-appb-000006
Figure PCTCN2017089196-appb-000007
注：正反向引物池是将表2所述的所有相应引物等比混合得到的。
扩增条件如下：
步骤1 98℃2min
步骤2 98℃10s
步骤3 58℃2min
步骤4 72℃30s
步骤5 98℃10s
步骤6 68℃1min
步骤7 重复步骤5-6，25个循环
步骤8 72℃5min
将每一个样品的扩增产物分别用1.8倍体积的anxygen beads进行纯化，最后溶于37ul TE中，备用。
3.文库制备
将上步获得的各样品的纯化产物分别依次进行USER酶切和末端修复加“A”、接头连接和PCR扩增，具体如下：
1)USER酶切和末端修复加“A”的体系为：
Figure PCTCN2017089196-appb-000008
其中，USER酶切和末端修复加“A”的反应程序为：20℃30min；65℃，30min。
2)接头连接的反应体系为：
Figure PCTCN2017089196-appb-000009
Figure PCTCN2017089196-appb-000010
接头连接的反应程序为：25℃15min；4℃10min。
然后，将得到的产物用1.2倍体积的anxygen beads进行纯化，最后溶于21TE中，备用。
3)PCR扩增
针对上步得到的各样品的连接产物，分别按照下表中的反应体系进行PCR扩增：
Figure PCTCN2017089196-appb-000011
其中，
正向通用引物的序列为(5’-3’)：
TGTGAGCCAAGGAGTTGAAGTGGCGCATTGTCTTCCTAAGACCGCTTGGCCTCCGACTT(SEQ ID NO：37)；
反向通用引物的序列为(5’-3’)：GAACGACATGGCTACGATCCGACTTGG(SEQ ID NO：38)。
PCR扩增程序如下：
Figure PCTCN2017089196-appb-000012
将得到的产物用1.2倍体积的anxygen beads进行纯化，最后溶于21TE中，即得目的文库。然后对得到的产物进行2100进行片段分析和qubit定量，结果见图5和表3。
其中，如图5所示，1-5为阳性样本，6-10位阴性样本。
表3：实验起始用量和文库产量
Figure PCTCN2017089196-appb-000013
4.测序上机
文库质检合格后进行上机测序，采用BGI seq500测序平台，测序类型为PE50BP。
5.数据分析
将得到的测序结果进行数据分析，具体包括：对下机测序reads进行过滤，过滤掉碱基质量小于10的reads，保证用于分析碱基频率的数据都是高质量的；使用bwa(V0.7.7-r441)比对软件将过滤后的clean reads比对回人基因组参考序列HG19(GRCH37)；根据比对结果计算出在待检测位点4个碱基ATCG的覆盖深度，并根据碱基深度得到最可能变异位点及其频率，测序错误及频率等情况；根据位点和引物所在位置的区间的GC分布情况及可能的测序错误频率，进行GC bias校正；使用目前最准确的变异检测工具GATK(V3.6)检测上述得到的变异位点，并使用相应突变数据库进行过滤和注释。
结果，发明人发现，利用本发明PCR引物进行多重PCR扩增和文库构建，可以准确检测低至1％的突变，具体地：在10例样本检测中，5例阳性样本均检测为阳性，检测到的突变频率分别为5.3％、7.2％、3.5％、5.6％、6.2％，5例阴性样本均检测为阴性，检测到的突变频率分别为0.3％、0.1％、0.2％、0.2％、0.1％(如表5)。并且，本发明的方法特异性好，比对率在90-94％之间，捕获效率在92-95％之间；覆盖度为100％(如表4)；均一性好，0.1X平均深度大于94％(见表4，图6)；稳定性好，不同样本之间差异小(见图7)。
表4：PE50+10下机数据
Figure PCTCN2017089196-appb-000014
Figure PCTCN2017089196-appb-000015
*均一性：大于0.1X平均reads数的扩增子数量(18/19＝94.7％)。
表5：突变检测准确性
Figure PCTCN2017089196-appb-000016
此外，发明人还进行了一系列实验，并发现本发明的PCR引物对的适用范围不限于cfDNA样本，还可以广泛应用于genomic DNA、FFPE、尿液DNA、fresh frozen等样本，也不仅限于构建BGI-Seq文库，也可以构建其他平台文库如illumina、proton。
工业实用性
本发明的PCR引物对，能够有效地用于待测DNA样品的PCR扩增，并且能有效降低PCR扩增过程中的GC偏向性，提高扩增特异性，且尤其适于作为多重PCR扩增的引物。
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 (17)

  1. 一种PCR引物对，其特征在于，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，
    所述第一特异性序列和所述第一随机序列之间连接有预定酶切位点，
    所述第二特异性序列和所述第二随机序列之间连接有预定酶切位点，且所述PCR引物对的靶序列不包含所述预定酶切位点。
  2. 根据权利要求1所述的PCR引物对，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PCR引物对，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15-45bp，所述第一特异性序列和所述第二特异性序列的长度为15-30bp。
  4. 根据权利要求1所述的PCR引物对，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PCR引物对，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PCR引物对，其特征在于，所述预定酶切位点为USER酶的酶切位点碱基U。
  7. 一种PCR扩增试剂盒，其特征在于，包含权利要求1-6任一项所述的PCR引物对。
  8. 一种PCR扩增方法，其特征在于，利用权利要求1-6任一项所述的PCR引物对或者权利要求7所述的PCR扩增试剂盒进行所述PCR扩增。
  9. 根据权利要求8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，使所述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。
  10. 根据权利要求9所述的方法，其特征在于，所述方法的扩增反应程序如下：
    Figure PCTCN2017089196-appb-100001
    Figure PCTCN2017089196-appb-100002
  11. 一种制备DNA文库的方法，其特征在于，包括以下步骤：
    (1)根据权利要求8-10任一项所述的方法，将待测DNA样品进行第一PCR扩增，以便获得包含类环状物的第一PCR扩增产物，其中，所述第一特异性序列和所述第一随机序列之间连接有预定酶切位点，所述第二特异性序列和所述第二随机序列之间连接有预定酶切位点，且所述PCR引物对的靶序列不包含所述预定酶切位点，所述类环状物的5’端和3’端未连接，采用的PCR引物对的第一引物和第二引物的5’末端和3’末端的第1-5个碱基经过硫代修饰；以及
    (2)利用所述预定酶切位点对应的酶，将所述第一PCR扩增产物进行酶切，并基于酶切产物进行文库制备，以便获得目的DNA文库。
  12. 根据权利要求11所述的方法，其特征在于，所述预定酶切位点为USER酶的酶切位点碱基U。
  13. 根据权利要求12所述的方法，其特征在于，按照如下的方法进行步骤(2)：
    将所述包含类环状物的第一PCR扩增产物进行所述酶切以及末端修复加A，以便获得末端包含碱基A的酶切产物；
    将所述末端包含碱基A的酶切产物进行接头连接，以便获得连接产物，所述连接产物为线性DNA；
    将所述连接产物进行第二PCR扩增，以便获得第二PCR扩增产物，所述第二PCR扩增产物构成线性DNA文库，其中所述第二PCR扩增产物采用测序引物进行，所述测序引物与所述接头序列互补。
  14. 根据权利要求13所述的方法，其特征在于，利用USER酶、T4多聚核酸激酶、T4 DNA聚合酶和Klenow片段进行所述酶切以及末端修复加A。
  15. 根据权利要求12所述的方法，其特征在于，按照如下的方法进行步骤(2)：
    将所述包含类环状物的第一PCR扩增产物进行所述酶切，以便获得包含类环状物的酶切产物；
    利用连接酶将所述包含类环状物的酶切产物进行连接反应，以便使所述类环状物的5’端和3’端连接成环；
    去除连接产物中的线性DNA，以便获得环状DNA混合物；以及
    将所述环状DNA混合物进行第三PCR扩增，以便获得第三PCR扩增产物，所述第三PCR扩增产物构成环状DNA文库，其中，所述第三PCR扩增的正向引物的3’端序列与所述第一随机序列反向互补，所述第三PCR扩增的反向引物的3’端序列与所述第二随机序列反向互补。
  16. 根据权利要求15所述的方法，其特征在于，利用线性消化反应去除线性DNA。
  17. 根据权利要求15所述的方法，其特征在于，在进行所述第三PCR扩增之前，进一步包括对所述环状DNA混合物进行纯化的步骤。
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