阅读说明：本技术 一种折叠引物的pcr扩增方法 (A kind of PCR amplification method folding primer ) 是由 黄天谊 于 2019-08-30 设计创作，主要内容包括：本发明公开了一种折叠引物的PCR扩增方法,所述PCR扩增有预退火PCR扩增和直接分级退火PCR扩增两种扩增方法。本发明能避免引物之间的竞争,实现在一个试管内互不干扰地进行多重反应；尤其是预退火PCR在无酶环境下先行反复分级退火,使所有靶序列都与对应引物充分退火,在反复中错配的引物可重新选择与靶序列退火；后续PCR保证靶序列的特异扩增始终占据绝对优势,有效地解决传统PCR极易产生非特异扩增、在多重反应中难于扩增低拷贝靶序列等弊病,提高了检出SNP等单碱基变异,同时检出多种靶核酸,特别是低拷贝靶核酸的特异性和敏感性,具有超灵敏、高特异、高通量的优点。(The invention discloses a kind of PCR amplification method for folding primer, the PCR amplification has preannealing PCR amplification and directly classification annealing two kinds of amplification methods of PCR amplification.The invention can avoid the competition between primer, realization carries out multiple reaction in a test tube without interfering with each other；Especially preannealing PCR classification annealing repeatedly in advance under no enzyme environment, makes all target sequences all sufficiently anneal with corresponding primer, can reselect in the primer of middle mispairing repeatedly and target sequence is annealed；Subsequent PCR guarantees that the specific amplified of target sequence occupies absolute predominance always, normal PCR is efficiently solved easily to generate non-specific amplification, be difficult to expand the disadvantages such as low-copy target sequence in multiple reaction, improve the single bases variation such as detection SNP, a variety of target nucleic acids are detected simultaneously, the especially specificity and sensibility of low-copy target nucleic acid, has the advantages that hypersensitive, Gao Teyi, high throughput.)

1. a kind of PCR amplification method for folding primer, it is characterised in that: the folding primer has triple move back in PCR reaction Fiery temperature and temperature detect switch (TDS) function；The PCR amplification method for folding primer has preannealing PCR and directly classification annealing PCR two Kind amplification method, two kinds of amplification methods are all made of primer, enzyme separation composite reagent mode, prepare No. 1 composite reagent and No. 2 respectively Composite reagent prepares DNA profiling with stencil buffers liquid.

2. a kind of PCR amplification method for folding primer according to claim 1, which is characterized in that the preannealing PCR expands Increasing method the following steps are included:

1. No. 2 composite reagents of 5 μ L and the template DNA of 5 μ L are added in a PCR pipe, upper machine presses preannealing program after centrifugation Classification annealing 2~5 times repeatedly in advance, 5~10min of time-consuming；

2. preannealing terminates, reaction tube is moved into room temperature, 5 μ L of No. 1 reagent is added and forms complete reaction system, upper machine is by default pre- Annealing PCR program successive reaction 55~65 recycle, and reaction was completed after 45~50min of time-consuming, obtains amplified production.

3. it is according to claim 2 it is a kind of fold primer PCR amplification method, it is characterised in that: step 1., it is described pre- Cycle of annealing are as follows: 1 genome melting temperature (86~95 DEG C/60~90s), >=2 initial annealing temperatures of primer are (70~50 DEG C/5~10s), 1~2 primer fold temperature is (45~65 DEG C/5~10s)；Described refers to repeatedly from >=highest annealing temperature Spent between minimum folding temperature repeatedly；First repeatedly since genome melting temperature in short-term, starts temperature repeatedly every time later Degree successively successively decreases from highest annealing temperature, until minimum annealing temperature and minimum folding temperature.

4. it is according to claim 2 it is a kind of fold primer PCR amplification method, it is characterised in that: step 2., it is described pre- It anneals PCR program are as follows: the 1st circulation of no melting temperature: since≤minimum folding temperature (such as 45 DEG C), 1~2 annealing temperature It is transitioned into 72~75 DEG C of the first chain extension temperature；Subsequent is 0~2 classification anneal cycles group, totally 0~20 circulation；With 1 height Warm anneal cycles group, totally 40~60 recycle；The classification anneal cycles are by amplicon melting temperature, >=1 initial annealing temperature Degree and elongating temperature composition；The high annealing circulation is by amplicon or genome melting temperature, high temperature anneal temperature and extension Temperature composition.

5. a kind of PCR amplification method for folding primer according to claim 1, it is characterised in that: the folding primer 3 ' hold custom primer part, when necessary its ad hoc 1-2 deoxyinosine in 3 ' end 2-4 and/or base mismatch/or degeneracy alkali Base；5 ' end special designing the primer portions for folding primer, by adding the base composition complementary with its 3 ' end, when necessary 5 ' The ad hoc 1-2 deoxyinosine in end 3-9 replaces complementary base, to avoid continuous >=3 CG complementary bases.

6. a kind of PCR amplification method for folding primer according to claim 1, it is characterised in that: No. 1 composite reagent Main component is MgCl₂, Taq enzyme, KCl and Tris-HCl.

7. a kind of PCR amplification method for folding primer according to claim 1, it is characterised in that: No. 2 composite reagents Main component is NH₂SO₄, 4dNTP, Tris-HCl and at least 1 pair folding primer.

8. a kind of PCR amplification method for folding primer according to claim 1, it is characterised in that: the stencil buffers liquid For 20mMTris-HCl (i.e. 1.0XTBF)；No. 2 reagents are directly added into when preparing filter paper blood template DNA using 1.0XTBF, such as The template DNA that fruit is extracted with kit then first plus after equivalent 2.0XTBF mixing adds No. 2 reagents.

9. a kind of PCR amplification method for folding primer according to claim 1, which is characterized in that the direct classification is moved back Fiery PCR amplification method the following steps are included:

(1) No. 2 composite reagents of 5 μ L and the template DNA of 5 μ L are added in a PCR pipe；

(2) No. 1 reagent of 5 μ L, upper machine after brief centrifugation, by directly classification annealing PCR program, successive reaction 55~65 is added 45~50min of a circulation time-consuming, reaction was completed, obtains amplified production.

10. a kind of PCR amplification method for folding primer according to claim 9, it is characterised in that: the direct classification is moved back Fiery PCR program are as follows: initial denaturation (84~95 DEG C/60~90s of genome unwinding), 1~3 genome unwinding are classified anneal cycles group Totally 2~12 circulations, totally 0~8 circulation, 1 high annealing circulation group are total for 0~2 amplicon unwinding classification anneal cycles group 40~58 circulations；Genome unwinding classification anneal cycles group by genome melting temperature (84~95 DEG C/05s), >=1 Initial annealing temperature (55~70 DEG C/5~10s) and (72~75 DEG C/5~10s) of elongating temperature compositions；The amplicon unwinding point Grade anneal cycles group by amplicon melting temperature (82~90 DEG C/05s), 1~2 initial annealing temperature (55~70 DEG C/5~ 10s) formed with (72~75 DEG C/5~10s) of elongating temperature；The high annealing circulation group is by amplicon or genome unwinding temperature Spend (82~95 DEG C/05s), high temperature anneal temperature (60~72 DEG C/10~20s) and elongating temperature (72~75 DEG C/5~10s) group At.

Technical field

The invention belongs to technical field of molecular biology more particularly to a kind of PCR amplification methods for folding primer.

Background technique

It is big that polymerase chain reaction (PCR Mullis.k 1985) appearance 30 for many years has become molecular biology three One of key technology (PCR, molecular cloning, nucleic acid sequence analysis-sequencing).It is widely used in the every field of life science；Mesh Preceding nucleic acid amplification technologies have become nearly all research for being related to biology and application field (including biology and genetic engineering, it is clinical With preventive medicine, agriculture woods, is herded, the biological related industry such as fishing and food, drug) molecular biology routine techniques.It uses PCR nucleic acid amplification technologies are expanded from various samples, are obtained, detecting interested nucleic acid sequence, are various nucleic acid (gene) Further analysis, research and application provide quick reliable and economic means.

PCR is a kind of method of external enzyme' s catalysis specific DNA fragment, is that efficiency is most in existing various nucleic acid amplification methods High, most widely used basic nucleic acid amplification method.The basic principle is that: be primer annealing complementary with target (target) nucleic acid with it is new The synthesis (extension) of raw complementary strand: in solution (biochemistry) environment of appropriate nucleic acid synthesis (containing required archaeal dna polymerase, extremely Few a pair of Oligonucleolide primers, target DNA (template), Mg⁺, 4dNTP and buffer system etc. that is appropriate and stablizing pH), setting Under conditions of nucleic acid denaturation, annealing and extending 3 temperature cycles variations, the template target DNA when temperature rises to denaturation temperature It is denaturalized (unwinding), primer is annealed in conjunction with complementary target DNA when being down to annealing temperature, during elongating temperature, is polymerize by DNA Enzyme catalysis, primer extend since its 3 end using target DNA as template along 5 ' -3 ' directions, and synthesis one is complete with target DNA Complete complementary new nucleotide chain；When second circulation, newly synthesized chain extends as the template of reverse primer from another direction, Until synthesizing new chain to previous primer starting, to define the design length of new chain, each cyclic amplification product is with index Multiplication loops back and forth like this after 30 circulations, and completely same amplified production can reach hundreds of thousands of to 1,000,000 times.Often A circulation all synthesizes extension by what the special annealing of primer and target sequence caused nascent strand, it is seen that the annealing of primer and target sequence is A step of most critical in nucleic acid amplification reaction.

Although round pcr has above-mentioned great advantage, but there is also intrinsic easily generation dimers etc. for normal PCR Non-specific amplification, amplified production easily pollute and advantage pcr is covered low-copy target sequence amplification three and lacked greatly when multiple reaction It falls into, seriously affects the accuracy of analysis.In various biology laboratories, it by most convenient and it is efficient in a manner of for us obtain nothing The significant nucleic acid of number, while also generating the wrong product for being largely enough failure test result.Especially in multiple reaction, In Same reaction system inner primer efficiency is irregular, the situation of template abundance height great disparity frequent occurrence under conditions of, can not keep away The competition exempted from and the mispairing that takes place frequently cause the multiple reaction of normal PCR often to end in failure.

Up to the present, to adapt to various needs such as molecular biology, life science and bioengineering, developed Tens of kinds of PCR amplification methods, including by template of DNA and using RNA as template two large divisions；Wherein using RNA as template majority It is related with reverse transcription PCR, in terms of being mainly used in basic research: such as amplifying target genes, clone gene, gene order-checking, base Because of function and expression regulation and mutagenicity study etc.；It is also used for detection, diagnosis and the research of fractionated viral (RNA virus) cause of disease. Biological genus and species (group) are widely used in using DNA as the PCR of template, type (genotype) includes mutated gene, transgenosis kind, type Detection, identification and research；Medicine (clinical and prevention), the inspection of environment infectious disease pathogens microorganism (virus, bacterium, helminth) Out, it identifies and studies；The detection, identification and research of hereditary disease lesion (mutation) gene；And it application in terms of medical jurisprudence and grinds Study carefully.But its PCR amplification part of all these PCR methods: including design of primers, reagent combination, test procedure expands mode It is all still the mode of normal PCR；So all there is the interference of mispairing and competition, especially in multiple reaction, can not overcome competing It strives, non-specific amplification and be difficult to expand the disadvantage of low-copy target sequence.

To solve above-mentioned problem existing for normal PCR over nearly more than 20 years, some improved PCR amplifications have also been developed； Such as technical literature discloses a kind of annealing control primer for " Annealing control primer and its uses " It is that 2-15 universal base (deoxyinosine etc.) one adjuster of composition of insertion draws for controlling between the end of primer 3 ' and 5 ' ends The annealing of object, it is said that being applicable to all spectra of nucleic acid amplification, non-specific amplification can be effectively inhibited and improving amplification and inspection The specificity of target nucleic acid out；But exclusion competition and detection ultra-drop target nucleic acid are not referred to, is not mentioned in multiple reaction especially Detect the ability of low-copy target nucleic acid.

Currently, also there is document to disclose PCR amplification, such as:

1, patent application CN201811475873.2 discloses a kind of PCR amplification method, includes the following steps: sample point Class label, reaction table production, example reaction, sample data processing and etc.；But the patent application can not allow primer to carry out Classification annealing, the primer of mispairing non-target sequences have no chance to reselect target sequence, cannot efficiently solve normal PCR and commonly draw Object easily generates non-specific amplification, is difficult to expand the disadvantages such as low-copy target sequence in multiple reaction, and primer cannot be allowed accurate, steady Fixed carry out multiple reaction.

2, patent application CN201711011855.4 discloses a kind of PCR amplification method, wherein the primer is sulfydryl Change the primer of modification, the PCR amplification method yield of the invention is high, sensitivity significantly increases；But the patent application can not allow Primer carries out classification annealing, and the primer of mispairing non-target sequences has no chance to reselect target sequence, cannot efficiently solve tradition PCR general primer easily generates non-specific amplification, is difficult to expand the disadvantages such as low-copy target sequence in multiple reaction.

3, patent application CN201510044665.7, this is our a kind of folding primer and its PCR disclosed in 2015 It is different to fold primer by setting every (to) using the amplification method for the single composite reagent that magnesium, enzyme separate for amplification method Annealing and folding temperature, complementary self-isolation of annealing in 3 ' 5 ' ends of primer when folding temperature lower than it；Prevent primer and non-target Mispairing annealing between sequence, effectively excludes the competition between primer, realizes that multipair primer is without interfering with each other in a test tube First after annealing, 4 kinds of plasmodiums of primary amplification while detection and identification.In practice behind, we gradually have found magnesium, enzyme separation Single composite reagent and its traditional amplification method could not give full play to the superior function for folding primer, single composite reagent mode Also it is unfavorable for saving and uses, folds the structure design aspect of primer there is also some defects, influence its expanding effect and do not reach To optimum state！We carry out a series of innovative designs and Optimal improvements test to primer PCR is folded regarding to the issue above, finally It develops sensitiveer, more special, more stable a new generation and folds primer PCR.

Summary of the invention

The present invention is in order to solve the above technical problems, provide a kind of PCR amplification method for folding primer.Folding primer used Have the function of triple annealing temperature and temperature detect switch (TDS), and the folding primer is applied to polymerase chain reaction (i.e. folding primer PCR, but not limited to this) two kinds of PCR amplification methods: preannealing PCR and directly classification anneal PCR amplification method.Pass through setting Every (to) folds the different annealing of primer and folds temperature, changes control primer fold Self-Closing or solution using annealing temperature Chain opening carries out classification annealing, avoids the competition between primer, realizes that multipair primer carries out without interfering with each other in a test tube Multiple reaction.The preannealing PCR amplification method especially created, the classification annealing repeatedly in advance under ad hoc no enzyme environment, not only Even if singly copy target sequence has the annealing complementary with corresponding primer of abundant chance to all target sequences；And the primer of mispairing is in repeatedly Also de- chain is easy to reselect and target sequence annealing；In the absence of target sequence, the complementary annealing in the end of primer 3 ' 5 ' itself fold and Closing fundamentally prevents the mispairing between primer and any non-target sequences；In addition to protecting in reaction mixture at the end of preannealing It holds with outside the primer of the special annealed condition of target sequence, all free primer self-annealings fold static.No. 1 reagent is added at this time (containing Taq enzyme), PCR reacts since room temperature to elongating temperature, and the primer only specifically annealed with target sequence is extending, and expands The first chain increased can only be the complementary strand of target sequence；Subsequent PCR limits amplicon as amplification using amplicon unwinding circulation Template further consolidates preannealing specific amplified achievement, to guarantee the specific amplified of target sequence with occupying absolute predominance always Position.Normal PCR general primer is efficiently solved easily to generate non-specific amplification, be difficult to expand low-copy target sequence in multiple reaction Column and amplified production such as easily pollute at the disadvantages；Therefore the specificity and sensitivity of the single bases variation such as detection SNP are greatly strengthened Property, be conducive to expand and detect micro cause of disease DNA in sample, be particularly conducive to the target of low copy number in detection mixed infection sample Nucleic acid.

In order to reach purpose described above, the invention adopts the following technical scheme:

A kind of PCR amplification method folding primer, the folding primer have triple annealing temperature and temperature in PCR reaction Control switching function；The PCR amplification method for folding primer has preannealing PCR and directly classification annealing two kinds of amplification methods of PCR, Two kinds of amplification methods are all made of primer, enzyme separation composite reagent mode, prepare No. 1 composite reagent and No. 2 composite reagents respectively, use Stencil buffers liquid prepares DNA profiling.

Further, the preannealing PCR amplification method the following steps are included:

1. No. 2 composite reagents of 5 μ L and the template DNA of 5 μ L are added in a PCR pipe, upper machine presses preannealing after centrifugation Program is classified annealing 2~5 times repeatedly in advance, 5~10min of time-consuming；

2. preannealing terminates, reaction tube is moved into room temperature, 5 μ L of No. 1 reagent is added and forms complete reaction system, upper machine is by pre- If preannealing PCR program successive reaction 55~65 circulations, reaction was completed after 45~50min of time-consuming, obtains amplified production.

Further, step 1., the preannealing program are as follows: 1 genome melting temperature (86~95 DEG C/60~ 90s), >=2 the initial annealing temperature of primer is (70~50 DEG C/5~10s), and 1~2 primer fold temperature is (45~65 DEG C/5 ~10s)；It is described refer to repeatedly from >=highest annealing temperature between minimum folding temperature repeatedly；First repeatedly from base in short-term Because a group melting temperature starts, start temperature is successively successively decreased from highest annealing temperature repeatedly every time later, until minimum annealing temperature With minimum folding temperature.

Further, step 2., the preannealing PCR program are as follows: the 1st circulation of no melting temperature: from≤minimum folding Folded temperature (such as 45 DEG C) start, and 1~2 annealing temperature is transitioned into 72~75 DEG C of the first chain extension temperature；Subsequent is 0~2 point Grade anneal cycles group, totally 0~20 recycles；With 1 high annealing circulation group, totally 40~60 are recycled；The classification annealing follows Ring is by amplicon melting temperature, >=1 initial annealing temperature and elongating temperature composition；The high annealing circulation is by amplicon Or genome melting temperature, high temperature anneal temperature and elongating temperature composition.

Further, custom primer parts are held in the 3 ' of the folding primer, when necessary the ad hoc 1-2 in its 3 ' end 2-4 A deoxyinosine and/or base mismatch/or degeneracy base；5 ' end special designing the primer portions for folding primer, by adding The base composition complementary with its 3 ' end, 5 ' end 3-9, ad hoc 1-2 deoxyinosine replaces complementary base when necessary, with Avoid continuous >=3 CG complementary bases.

Further, No. 1 composite reagent main component is MgCl₂, Taq enzyme, KCl and Tris-HCl.

Further, No. 2 composite reagent main components are NH₂SO₄, 4dNTP, Tris-HCl and at least 1 pair folding draw Object.

Further, the stencil buffers liquid is 20mMTris-HCl (i.e. 1.0XTBF)；Filter paper is prepared using 1.0XTBF No. 2 reagents are directly added into when blood sample template DNA, if the template DNA extracted with kit first plus after equivalent 2.0XTBF mixing Add No. 2 reagents.

Further, it is described directly classification annealing PCR amplification method the following steps are included:

(1) No. 2 composite reagents of 5 μ L and the template DNA of 5 μ L are added in a PCR pipe；

(2) No. 1 reagent of 5 μ L, upper machine after brief centrifugation, by directly classification annealing PCR program, successive reaction 55 is added ~65 circulation 45~50min of time-consuming, reaction was completed, obtains amplified production.

Further, the directly classification annealing PCR program are as follows: initial denaturation (genome unwinding 84~95 DEG C/60~ 90s), totally 2~12 circulations, 0~2 amplicon unwinding are classified anneal cycles to 1~3 genome unwinding classification anneal cycles group Totally 0~8 circulation, 1 high annealing circulation group recycle for 40~58 totally group；Genome unwinding classification anneal cycles group by Genome melting temperature (84~95 DEG C/05s), >=1 initial annealing temperature (55~70 DEG C/5~10s) and elongating temperature (72 ~75 DEG C/5~10s) composition；Amplicon unwinding classification anneal cycles group by amplicon melting temperature (82~90 DEG C/ 05s), 1~2 initial annealing temperature (55~70 DEG C/5~10s) and (72~75 DEG C/5~10s) of elongating temperature compositions；It is described High annealing circulation group is by amplicon or genome melting temperature (82~95 DEG C/05s), high temperature anneal temperature (60~72 DEG C/10 ~20s) and (72~75 DEG C/5~10s) of elongating temperature compositions.

Further, the amplified production analyze and identify can be used agarose gel electrophoresis separation it is big according to amplified fragments Small identification；Or the similar or similar of the molecular weight comparison database storage of amplified fragments is measured using efficient mass spectrum inspection (HPMS) Population data is identified；It can be identified using microarray or low density chip；It can also be expanded with determining nucleic acid sequence technical appraisement Increase segment section.

It is herein described fold patent that primer is application in inventor 2015 (patent name: a kind of folding primer and its PCR amplification method, application number: CN201510044665.7) in the folding primer mentioned, the folding primer sequence are as follows: 5 ' CCGTGATGTGGAGAATGTTTTGCATCACIT 3’。

In the application, the circulation is unwinding, annealing, the circulation for extending three steps, and annealing step can contain multiple annealing temperature Degree；Annealing is divided into initial annealing, high annealing；The initial annealing temperature can in the circulation (group) or between circulation (group) according to Be incremented by the sequence perhaps successively decreased carry out classification annealing or in circulation (group) or between circulation (group) according to being alternately incremented by or The sequence successively decreased carries out classification annealing, and the time of each annealing temperature processing is 1~15s；The initial annealing temperature At 45~70 DEG C；During multiplexed PCR amplification, the initial annealing temperature between each pair of primer differs at least 3~5 DEG C；It is described Folding primer, folding temperature is 43~68 DEG C, and in cyclic process, the folding temperature of each pair of primer is lower than initial annealing At least 1~2 DEG C of temperature；The folding primer during PCR amplification, high temperature anneal temperature be 60~72 DEG C, annealing when Between be 5~20s；The elongating temperature is 72~75 DEG C, and extension of time is 5~20s.

The high annealing can be made annealing treatment using single high temperature, can also use 2~3 high annealing temperature Degree carries out being incremented by classification annealing.

The folding primer has the function of that triple annealing temperature and temperature control locking or unwinding are open in PCR reaction, including 3 ' end custom primer parts and 5 ' end special designing primer portions；3 ' end custom primer the parts for folding primer are according to need The specific site for detecting and/or identifying designs the one section nucleotide sequence complementary with target gene, and composition and length decision are drawn The upper limit of object and target sequence annealing temperature, referred to as primer it is initial annealing or low-temperature anneal temperature, when necessary its 3 ' end the 2nd~ 4 ad hoc 1~2 deoxyinosines and/or base mismatch/or degeneracy base；5 ' end special designing the primers for folding primer The base composition complementary with its 3 ' end, special designing primer portion and 3 ' ends mainly are added by 5 ' ends of custom primer part in part Complementary series is to add 7~19 and 3 ' end complementary bases to the 8th~20 base from the 2nd~4 base, the complementary base Composition and length determine the temperature of primer fold self-annealing, which is known as folding temperature up to 100% temperature, Determine the lower limit of initial annealing temperature；5 ' end the 3rd~9, ad hoc 1~2 deoxyinosine replaces complementary base when necessary, And 5 ' ends the 1st it is then ad hoc be and the 1st bit mismatch base of 3 ' end；The base mismatch is one of C or G；The folding After at least two recycles, 5 ' ends of amplicon have generated the complementary series for folding primer overall length for folded primer PCR reaction；Hereafter Primer extension amplification mainly using amplicon as template, at this time fold primer overall length it is complementary with amplicon annealing, and be more than often Primer portion is advised, the annealing temperature for folding primer will increase >=5 DEG C, referred to as high temperature anneal temperature automatically；The solution of the amplicon Chain temperature is 82~90 DEG C.

The basic principle for folding primer is to fold or be overlapped (itself by the complementary annealing in temperature regulation primer 3 ' 5 ' end Or closing mutually) or unwinding (opening) control the annealing of primer Yu template target sequence, each item (to, group) primer is arranged different Annealing temperature and folding temperature carry out classification and anneal, in the reaction in the preannealing PCR amplification method especially innovated, in nothing Classification annealing repeatedly, can not only exclude the competition between primer under enzyme environment, while fight conventional polymeric enzyme chain reaction (PCR) warp It often results between the primer of non-specific amplification and primer or probe, and/or mispairing is annealed between primer and non-target sequences；Make to own Even if singly copy target sequence has the opportunity to sufficiently anneal with corresponding primer target sequence；The primer of accidental mispairing in repeatedly also easily Fall chain and reselect target sequence and specifically anneals；Non-specific amplification can be easily generated efficiently against normal PCR, in multiple reaction It is difficult to expand the disadvantages such as low-copy target sequence, to greatly improve the specificity of PCR, stability and amplification efficiency.Therefore, this hair It is bright to can be directly used for improving sleeve type PCR, multiplex PCR, Allele specific PCR, the polymerase chain reaction of quantitative fluorescent PCR and RNA RNA-PCR and reverse transcription PCR, the end cDNA rapid amplifying (RACE), differential display PCR (DD-PCR) etc. are various to be based on The Nucleic acid assays of PCR, defect that can be intrinsic efficiently against above-mentioned normal PCR improve specificity and amplification efficiency；Folding is drawn Object cooperate primer, enzyme separate composite reagent reaction system, can be used for developing high specificity and sensitivity can detect simultaneously a variety of cause of diseases/ The novel pre- tubulature PCR molecule diagnosis kit of lesion/mutant gene or SNP；It can also be applied to nucleic acid to extend, hybridize correlation The specificity that the technology design of folded target probe (such as in microarray) extends efficiently against Non-specific hybridization and enhancing, this Outside, in chip and high performance liquid chromatography (HPLC) the detection foranalysis of nucleic acids system such as nucleic acid mutation and SNP, folding primer PCR will Efficient, quick and high quality sample preparation is provided.

The improved nucleic acid amplification method of the present invention (folding primer PCR) folds the triple annealing temperature of primer by every, The each PCR test of flexible arrangement as needed carries out classification denaturation, annealing, extension.Can in duplicate circulation (group It is interior) classification annealing extension, denaturation, annealing can also be classified (between group) between different circulations and is extended；Both be more often It matches to obtain best expanding effect.

Since present invention employs above technical schemes, have the advantages that

(1) present invention is the basis of folding primer disclosed in inventor 2015 (patent No.: CN201510044665.7) Upper further research and development, a new generation released after improving fold primer and its PCR amplification method.It can autonomous Design be triple moves back except having Fiery temperature and temperature detect switch (TDS) function etc. fold outside all advantages of primer, eliminate the defect folded in the design of primer original, a new generation The complex situations that primer is suitable for the various different bases compositions of 3 ' terminal specific primers are folded, even if continuous >=3 C occur in 3 ' ends Or G, it folds primer and is also able to maintain the superior function of temperature detect switch (TDS) freely, it is competing that exclusion is preferably played especially in multiple reaction The effect for striving, preventing mispairing further improves the sensibility and specificity for folding primer PCR.

(2) present invention innovation is using the folding primer and preannealing PCR with triple annealing temperature and temperature detect switch (TDS) function Amplification method folds the different annealing of primer by setting every (to) and folds temperature, and both of which carries out classification annealing, The competition between primer is avoided, realization carries out multiple reaction in a test tube without interfering with each other；Especially preannealing PCR exists Classification annealing repeatedly in advance, makes all target sequences all sufficiently anneal with corresponding primer under no enzyme environment, in drawing for middle mispairing repeatedly Object can be reselected anneals with target sequence；The composite reagent mode separated using primer, enzyme, in the environment of no enzyme, folding is drawn Object and the sufficiently annealing repeatedly in advance of template DNA target sequence, are conducive to increase low-copy target sequence and the meeting of corresponding primer annealing machine, together When make the primer of mispairing non-target sequences have an opportunity to reselect target sequence, fundamentally prevent between primer and any non-target sequences Mispairing annealing；Subsequent PCR amplicon unwinding circulation consolidates the achievement of preannealing, and guarantee makes the specific amplified of target sequence always Superiority is occupied, normal PCR general primer is efficiently solved and easily generates non-specific amplification, is difficult to expand in multiple reaction The disadvantages such as low-copy target sequence；Therefore the specificity and sensibility for greatly strengthening the single bases variation such as detection SNP, detect simultaneously The specificity and sensibility of a variety of target nucleic acids, especially low-copy target nucleic acid are conducive to expand and detect micro cause of disease in sample DNA。

(3) the preannealing PCR amplification method innovated of the present invention, by setting every (to) fold the different annealing of primer and Folding temperature, when folding temperature lower than it, the complementation of the end of primer 3 ' 5 ' is annealed and self-isolation, is carried out repeatedly under no enzyme environment Classification annealing, not only eliminate the competition between primer, but also each pair of primer is made to have the opportunity to sufficiently anneal with target sequence, it is special Be not conducive to carry out multiple reaction without interfering with each other in a test tube, multiplex PCR is enable to detect and identify that simultaneously template is rich Spend all target sequences to differ greatly.

(4) the preannealing PCR amplification method that the present invention innovates, due to the composite reagent mode separated using primer, enzyme, In In the environment of enzyme, primer and the sufficiently annealing repeatedly in advance of template DNA target sequence are folded, subsequent PCR reaction is warm from folding Degree starts, without traditional thermal starting mode, without using more expensive hot start Taq polymerase.In addition, the primer of innovation, Enzyme separates composite reagent mode, and No. 1 reagent therein is actually a kind of efficient common reagent；Be conducive to develop hypersensitive, Folding primer multiplex PCR molecular diagnosis examination that is high special, high-throughput, inexpensive and quickly and easily detecting various Different Kinds of Pathogens Agent box, this will be real good and cheap quick practical kit.

(5) it is to fold the design and folding of primer that present invention a new generation, which folds another characteristic of primer PCR beyond tradition PCR, Folded primer PCR amplified reaction has broad Modulatory character.Site and length of traditional general primer on target sequence determine it Afterwards, its optimum annealing temperature has also substantially determined a kind of only selection；And the triple annealing temperature of every folding primer completely may be used With autonomous Design as needed, on the same site can there are many selection.The common simple PCR of 1 pair of primer or multipair primer Complicated PCR, once primer setting synthesis, suitable temperature cycles amplification program also determine that, only a kind of selection；And it rolls over No matter simple or multiplex PCR after folded primer synthesis, can be adjusted by setting preannealing program and subsequent PCR amplification program The precedence of each annealing temperature is saved, the amplification of the recurring number of length of time and each circulation group, each pair of primer of control is strong Degree, reaches optimum efficiency.

Specific embodiment

Specific embodiments of the present invention will be described in further detail below, but the invention is not limited to these realities Mode is applied, it is claimed to still fall within the claims in the present invention for any improvement or replacement on the present embodiment essence spirit Range.