CN1508258A - Polymerase chain reaction method for expanding ultrashort product and use thereof - Google Patents
Polymerase chain reaction method for expanding ultrashort product and use thereof Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 38
- 238000003752 polymerase chain reaction Methods 0.000 title claims description 21
- 238000006243 chemical reaction Methods 0.000 claims abstract description 41
- 230000004087 circulation Effects 0.000 claims abstract description 35
- 239000002299 complementary DNA Substances 0.000 claims abstract description 15
- 238000004519 manufacturing process Methods 0.000 claims description 54
- 239000000047 product Substances 0.000 claims description 39
- 238000004925 denaturation Methods 0.000 claims description 37
- 230000036425 denaturation Effects 0.000 claims description 37
- 108020004414 DNA Proteins 0.000 claims description 22
- 230000008859 change Effects 0.000 claims description 20
- 238000000137 annealing Methods 0.000 claims description 15
- 108020004635 Complementary DNA Proteins 0.000 claims description 13
- 238000010804 cDNA synthesis Methods 0.000 claims description 13
- 238000001514 detection method Methods 0.000 claims description 7
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 4
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 4
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- 239000012634 fragment Substances 0.000 abstract description 11
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- 239000002585 base Substances 0.000 description 30
- 238000002844 melting Methods 0.000 description 23
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- 238000013461 design Methods 0.000 description 11
- 102000004190 Enzymes Human genes 0.000 description 10
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- 230000003321 amplification Effects 0.000 description 10
- 238000003199 nucleic acid amplification method Methods 0.000 description 10
- 238000012360 testing method Methods 0.000 description 8
- 241000700721 Hepatitis B virus Species 0.000 description 4
- 239000003513 alkali Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 208000002672 hepatitis B Diseases 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 108010043137 Actomyosin Proteins 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
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- 238000010839 reverse transcription Methods 0.000 description 2
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Abstract
The invention relates to a polyase chain reaction method and application, providing an ultrashort product amplifying reaction method, the obtained PCR product is 24-150 basic groups in length. The method can become a universal applied PCR, and be applied to eliminating the pseudo-positive problem caused by the product pollution in during the course of detecting amplified fragment. And by contrasting with the reaction result of the template DNA under two denaturing temperatures 94-95 deg.C and 60-87 deg.C in the first 2 circulations, it can obviously differentiate the gene groups DNA and cDNA.
Description
Technical field
The present invention relates to Protocols in Molecular Biology, particularly relate to a kind of method and application thereof of polymerase chain reaction.
Technical background
The polymerase chain reaction is the method for a kind of specific DNA that increases efficiently, particularly is widely used in the clinical diagnosis in biomedical every field.The polymerase chain reaction has many parameters to comprise the reaction solution moiety, the reaction solution volume, and the sex change of reaction, annealing and elongating temperature time etc., wherein, PCR reaction amplified production length also is one of parameter.Amplified production length depends on selection of primers, has also just determined in case primer is determined the length of amplified production.See that from research always primer design is the key of PCR, but to concentrating on the length of analyzing primer itself, GC base ratio on the selection of primers, 3 ' end complementary base radix, aspects such as hairpin structure power, and the selection of the length of amplified production be placed on one very less important, the status of subordinate.Nearly all primer-design software does not all connect the preciseness of the length of amplified production and primer.Pcr amplification product length is usually in the 150-1000 base.Research about long range PCR (LD-PCR) in recent years increases day by day, and the PCR test kit that is fit to the long product of amplification also occurs successively, can amplify the long product of several thousand to several ten thousand bases under conditions suitable.With respect to the amplification research of long product, as if the amplification of short product studied still blank out.Point out that amplified production length is more than 150 bases so far various of PCR invention bar none about textbook and experiment guide.The primer-design software Oligo that is widely used is also the lower limit of amplified production 150 bases as default primer search area.These prove absolutely that existing round pcr has formed the general knowledge of product length greater than 150 bases, and Shang Weiyou studies at the PCR reaction less than 150 base products specially, and indicate the special technique effect that the short product of amplification brings.The present invention breaks through the prior art routine, process is to the calculating of the aspects such as melting temperature(Tm) of short amplified production, analyze and test, find that the short product of amplification can become a kind of PCR method of widespread usage, have the not available advantage of standard pcr, unique using value is being arranged aspect the detection amplified production pollution (carryover).
Summary of the invention
Technical problem to be solved
Technical problem to be solved by this invention provides a kind of polymerase chain reaction method and application thereof of the ultrashort product that increases, to break through the routine of amplified production length in the existing PCR method, overcome conventional PCR reaction and can not be used to distinguish the false-positive defective that amplified production pollutes.
Inventive concept
Principle of the present invention be the melting temperature(Tm) in view of a double chain DNA fragment depend primarily on its length but with its alkali base form with arrange relevant closely.When amplified production length less than 150 alkali bases during particularly less than 100 bases, its average melting temperature(Tm) reduces and significantly reduces with chain length, thereby the melting temperature(Tm) that is easy to find its amplified production of primer is between 68-85 ℃, and this amplified production can be finished denaturing step under than 68-85 ℃ of slightly high temperature.Our the all-cis preface of hepatitis B virus of alkali base surplus length 3000 is an example, (random device is in 1 of all-cis preface to have analyzed the melting temperature(Tm) of different lengths amplified production with the primer-design software random test, 201,401,601...... it is 30 that the position is got length respectively, 40,60...... each about 20 of amplified productions analyze) the results are shown in table one, statistic data shows when amplified production length is 30-100 alkali base, its average melting temperature(Tm) rises to 83 ℃ from 72 ℃, and minimum melting temperature(Tm) all is lower than 76 ℃.
Whether ultrashort product P CR method contains for discriminating and exists amplified production to pollute in the sample of genomic dna simple effective means is provided.The false positive that is caused by pollution is the big problem in the clinical PCR check, confirms that amplified production pollutes also trouble and will differentiate.Use ultrashort product P CR method,, the circulation of 2 initial at least conventional denaturation temperatures must be arranged then, just may obtain the target amplification product if do not exist amplified production to pollute in the sample; Otherwise, if in ultrashort product P CR total overall reaction process, all use under the situation of low denaturation temperature, also can obtain amplified production, interpret sample has to lag behind to be polluted.So in normal the detection, the controlled trial of doing a low denaturation temperature of PCR reaction whole process using just can judge.
Technical scheme
The present invention is based on conventional polymerase chain reaction method, in turn include the following steps: the template sex change; Primer annealing; Synthetic complementary dna chain is extended in archaeal dna polymerase catalysis down; And the circulation of abovementioned steps.Difference is that amplified reaction product length is the 24-150 base, and preferably in 24-100 base scope, the length range with best effect is the 40-70 base.Product D NA is less than 100 bases, and average melting temperature(Tm) significantly descends more obvious base length and the composition of depending on.
To decision, the present invention searches with manual method with primer-design software automated method search, the various collection of illustrative plates that also can utilize primer-design software to show the length of amplified production by primer.The length range of primer own is the 11-40 base, preferable range 18-30 base.
The reaction conditions of ultrashort amplified production PCR method sees Table 1.2 initial round-robin denaturation temperatures routinely, 68-96 ℃ of follow-up round-robin denaturation temperature, preferred 72-80 ℃.The annealing region of ultrashort amplified production PCR method is preferably implemented temperature 55-65 ℃ between 40-65 ℃, the elongating temperature scope is 45-72 ℃, preferably implements temperature 55-65 ℃.
The ultrashort amplified production PCR of table 1. reaction cycle condition
| General context | Preferable range | |
| Initial 2 or 3 circulation denaturation temperatures | ??92-96℃ | ????95-96℃ |
| Follow-up circulation denaturation temperature | ??68-96℃ | ????72-80℃ |
| Annealing temperature | ??40-65℃ | ????55-65℃ |
| Elongating temperature | ??45-72℃ | ????55-65℃ |
| The sex change time | 1-30 second | 1-5 second |
| Annealing time | 1-30 second | 1-5 second |
| The extension time | 1-60 second | 1-5 second |
Adopt ultrashort primer to carry out said polymerase chain reaction, make denaturation temperature can be lower than conventional 94-95 ℃.The template sample is carried out initial 2 round-robin reaction with 94-95 ℃ with 68-87 ℃ of two kinds of denaturation temperatures respectively, the template that can significantly distinguish in the DNA sample to be measured is that the fragment that long-chain DNA or previous amplified production bring is polluted, long-chain DNA can unwind at 94-95 ℃, and can't sex change under 68-87 ℃, all can amplify positive findings and pollute fragment 94-95 ℃ and 68-87 ℃ of two kinds of denaturation temperatures.Said ultrashort product polymerase chain reaction method has its unique application equally in the detection of distinguishing genomic dna and cDNA, design the corresponding primer of ultrashort product, and the template sample is carried out 2 PCR respectively:
(1) carries out initial 2 circulations with 94-95 ℃ of denaturation temperature, follow-up circulation 68-87 ℃ sex change;
(2) carry out initial 1 circulation with 94-95 ℃ of denaturation temperature, follow-up circulation 68-87 ℃ sex change.
Because genomic dna can unwind at 94-95 ℃, and can't sex change under 68-87 ℃, so genomic dna must have 2 high-temperature denatured circulations at least, just can completely increase; Only need 1 high-temperature denatured circulation can complete amplification and carry out complementary DNA (cDNA) that reverse transcription obtains with the single-minded anti-primer of gene.Be that genomic dna is positive in reaction (1), be negative in the reaction (2); CDNA all is positive in reaction (1) and (2).
Template DNA of the present invention is cDNA, utilizes cDNA to prepare test kit (the AdvantageRT for PCR kit of Clontech company) preparation, the Oligo that the primer provides for test kit (dT) by the total RNA of people's muscle tissue (Clontech company)
18, PCR volume 10 μ L, every pipe add 1 μ L, 1 μ g/1 λ cDNA.
It then is 0.5 μ M that PCR reaction of the present invention all adopts the Advantage of Clontech company 2 test kits, primer final concentration not to indicate.
Beneficial effect
Beneficial effect of the present invention is summarized as follows:
1: ultrashort amplified production PCR method can be combined into a step with the annealing and the extension of common PCR reaction cycle.
The amplified production of PCR of the present invention is the 24-100 base only, and this height of primer is the 12-40 base, so in fact only need to extend about the 12-80 base.Because the Taq polysaccharase is 22,37,55 or 70 ℃ extension speed is respectively per second 0.25,1.5,22 or 60 above Nucleotide, so in using general annealing region 40-65 of the present invention, can to tens seconds time, finish extension, needn't the accurate PCR method of image scale behind annealing steps, temperature be brought up to the optimum temperature scope of 70 ℃ of left and right sides Taq enzymes like that and kept for some time several.So each circulation of ultrashort amplified production PCR method only needs sex change and annealing to extend for two steps.
2: reaction fast.
The annealing of Standard PC R and extension step need 1 fen half usually altogether, and the sex change time is generally 30 seconds kinds, so that the higher amplified production of melting temperature(Tm) can fully unwind under 94-95 ℃.The melting temperature(Tm) of ultrashort amplified production of the present invention is extremely low, and denaturation temperature is controlled at than melting temperature(Tm) summary height, and under the condition that adopts reaction solution 10 microlitres, the sex change time of the present invention only was 1 second.Denaturation temperature of the present invention is between 70-85 ℃, and 50-65 ℃ of elongating temperature of annealing is identical with Standard PC R.The denaturation temperature of Standard PC R is between 94-95 ℃, so the denaturation temperature of the inventive method and the difference of annealing temperature are 5-35 ℃, and this value of Standard PC R is 30-45 ℃, is example with 55 ℃ of annealing temperatures, and each round-robin ramp time ratio Standard PC R of the present invention can save 25-60%.Because amplified production is shorter, the time of finishing extension also can foreshorten to several seconds or only utilize the ramp time to finish.In following test example, can both in 15 minutes, finish 30 round-robin PCR reactions, be below 1/4th of standard pcr required time.
3. the polysaccharase consumption can reduce greatly: the minimum dosage of PCR reactive polymeric enzyme need be considered 2 points, one, and along with the enzyme activity that carries out of reaction cycle slowly descends, the consumption of enzyme is allowed some leeway usually.They are two years old, concentration in reaction solution is per 100 microlitre 5U, every milligram of protein 20 of ratio vigor with the Taq enzyme, 000U and molecular weight are that 98KD can calculate, in the early stage of PCR reaction, the quantity of enzyme molecule is considerably beyond the amplified production molecular amounts, when PCR enters the later stage, under Standard PC R condition, enzyme molecule can catalytic product molecule number=enzyme catalysis speed (Nt/ second) * catalysis time (second) ÷ product length (Nt) in one extension period, and similarity condition can catalytic product molecule number improve in one extension period greatly for ultrashort product, and the requirement of enzyme concn is reduced greatly.In addition, the present invention improves the PCR speed of response greatly, and entire reaction course shortens, and temperature of reaction significantly descends, and the polysaccharase consumption can be decreased.
4. amplified production is shorter than 150 bases, makes PCR be reflected to be lower than 80 ℃ and even be lower than under 70 ℃ the condition and carry out, so the thermotolerance of archaeal dna polymerase is required to reduce, has expanded the range of choice of archaeal dna polymerase kind greatly.
5. enzyme molecule inactivation amount in reaction process reduces, and has also improved the stability of entire reaction course, can make PGR reaction cycle number surpass 30-35 common circulation.
6. specificity improves: amplified production is shorter than 150 bases, the product denaturation temperature is low, not only can be by primer and template order coupling, limit non-single-minded product with annealing temperature, stop normally unwinding of non-single-minded product by denaturation temperature again, make it not reach single-stranded structure and stop its further amplification.
Embodiment
Embodiment 1.
Verify ultrashort amplified production fragment possibility in design.
Utilizing the Oligo primer-design software is the right quantity and the right melting temperature(Tm) characteristic of typical primer of primer of example test amplified production length 40-1000 base with hepatitis B virus (HBV) complete genome sequence.
Table 2 shows the statistics that the melting temperature(Tm) of different lengths amplified production distributes, and data show that the average melting temperature(Tm) of amplified production increases with length when amplified production length during in the 200-1000 base, but variation tendency is more steady; When amplified production length during less than 200 bases, the average melting temperature(Tm) of amplified production reduces with length and obviously reduces, and minimum melting temperature(Tm) shows same trend.
Press the PCR reaction of table 2 design, the denaturation temperature of employing is 92-96 ℃ in initial 2 or 3 circulations, far above each the highest melting temperature(Tm), with 68-89 ℃ of sex change, also can satisfy and is higher than minimum melting temperature(Tm) requirement in follow-up circulation.Proof the solution of the present invention on design primer and product is feasible.
Table 2. with the full gene order of hepatitis B virus (HBV) be the unwinding of example different lengths amplified production/
Denaturation temperature
Table 2. with the full gene order of hepatitis B virus (HBV) be the unwinding of example different lengths amplified production/
Denaturation temperature
| Unwind/denaturation temperature (℃) | Amplified production chain length (base number) | ||||||||
| ??40 | ?60 | ??80 | ?100 | ??200 | ?400 | ??600 | ?800 | ??1000 | |
| Minimum melting temperature(Tm) | ??67.2 | ?71.3 | ??74.4 | ?76.3 | ??79.2 | ?82.0 | ??83.6 | ?84.3 | ??84.9 |
| The highest melting temperature(Tm) | ??82.5 | ?85.0 | ??88.8 | ?89.4 | ??91.7 | ?91.1 | ??90.2 | ?89.4 | ??88.8 |
| The highest lowest difference value | ??15.3 | ?13.7 | ??14.4 | ?13.1 | ??12.5 | ?12.3 | ??12.3 | ?11.2 | ??11.2 |
| Average melting temperature(Tm) | ??75.2 | ?79.1 | ??81.4 | ?82.8 | ??85.1 | ?86.0 | ??86.3 | ?86.5 | ??86.7 |
| Standard error | ??4.1 | ?4.1 | ??4.0 | ?3.7 | ??3.1 | ?2.6 | ??2.1 | ?1.7 | ??1.4 |
| Initial circulation denaturation temperature | ??92-96 | ?92-96 | ??92-96 | ?92-96 | ??92-96 | ?92-96 | ??92-96 | ?92-96 | ??92-96 |
| Follow-up circulation denaturation temperature | ??68 | ?72 | ??76 | ?78 | ??81 | ?83 | ??86 | ?88 | ??89 |
Embodiment 2.
Verify the primer feasibility in design of ultrashort product.
Utilize the Oligo primer-design software, with the hepatitis B virogene is example, test analysis the right frequency of occurrences of outstanding primer of the ultrashort amplified production that can increase, the result show the 30-100 base scope in any length, can search tens to hundreds of to having the primer of high preciseness, wherein, the melting temperature(Tm) of 30-80% amplified production is less than 80 ℃.Table 3 has outlined the analytical results to hepatitis B virogene.
The outstanding primer logarithm that table 3 finds in hepatitis B virogene
| Amplified production length | The amplified production melting temperature(Tm) | ||||
| ??＜70 | ?70.1-75 | ?75.1-80 | ?80.1-85 | ????＞85 | |
| ????31-40 | ????9 | ????41 | ????45 | ????5 | ????0 |
| ????41-50 | ????0 | ????18 | ????67 | ????14 | ????1 |
| ????51-60 | ????0 | ????1 | ????63 | ????34 | ????2 |
| ????61-70 | ????0 | ????1 | ????31 | ????66 | ????2 |
| ????71-80 | ????0 | ????0 | ????20 | ????66 | ????15 |
| ????81-90 | ????0 | ????0 | ????20 | ????71 | ????9 |
| ????91-100 | ????0 | ????0 | ????30 | ????55 | ????16 |
Embodiment 3.
Ultrashort product and design of primers example.
Utilize the Oligo primer-design software with people actomyosin gene complete genome sequence for example designs following primer.(design result sees Table 4)
Table 4. people actomyosin gene order designs the corresponding primer of ultrashort product for example
| The primer title | Sequence 5 '-3 ' | Primer length | Primer Tm (℃) | Product length | Product Tm (℃) |
| ?9A1?5′ ??9A1?3′ | ??CTT?TCG?TGT?AAA?TTA?TGT?AAT?GCA?A ?AAA?ATA?AAA?AAG?TAT?TAA?GGC?GAA?GAT | ????25 ????27 | ????65.8 ????66.0 | ????62 | ????67.9 |
| ?9A2?5′ ??9A2?3′ | ????TGG?ACA?TCC?GCA?AAG?ACC?T ????AGA?GAG?CAC?TGT?GTT?GGC?GT | ????19 ????20 | ????65.4 ????65.7 | ????41 | ????77.2 |
| ?9A3?5′ ??9A3?3′ | ????GGG?CAT?GGG?TCAG ????CGC?CCA?CAT?AGG?AAT | ????13 ????15 | ????47.9 ????52.1 | ????32 | ????76.1 |
| ?9A4?5′ ??9A4?3′ | ????GCG?CTC?GTC?GTC ????CGG?AGC?CGT?TG | ????12 ????11 | ????45.3 ????41.9 | ????25 | ????76.9 |
| ?9A5?5′ ??9A5?3′ | ????AAA?TGC?TTC?TAG?GCG?GAC?TAT?GA ????AAA?CAA?ATA?AAG?CCA?TGC?CAA?TC | ????23 ????23 | ????69.7 ????69.6 | ????103 | ????78.8 |
| ?9A6?5′ ??9A6?3′ | ???ACT?TAG?TTG?CGT?TAC?ACC?CTT?TCT ??CGT?TCC?AGT?TTT?TAA?ATC?CTG?AGT?C | ????24 ????25 | ????68.0 ????69.7 | ????144 | ????77.5 |
Embodiment 4.
Embodiment 3 designed primers are reacted carrying out ultrashort product P CR.
Reaction conditions is: 95 ℃ of sex change of elder generation 60 seconds, and 62 ℃ (for 9A1,9A2,9A5 and 9A6) or 45 ℃ (for 9A3 and 9A4) anneals and extends and carried out 2 or 3 circulations in 5 seconds; 68-82 ℃ of sex change is 5 seconds again, and 62 ℃ (for 9A1,9A2,9A5 and 9A6) or 45 ℃ (for 9A3 and 9A4) anneals and extended 5 seconds, totally 25 follow-up circulations.The results are shown in Table 5.
The ultrashort product P CR reaction result of table 5.
| Primer is right | Product length | Initial cycle number | Follow-up circulation denaturation temperature (℃) | ||||||||||
| ??68 | ??70 | ??72 | ??73.7 | ??74.9 | ??76.4 | ??78.1 | ??79.5 | ??80.5 | ??81.3 | ??82 | |||
| ??9A1 | ??62 | ????3 | ??± | ??+ | ??+ | ??+ | ??+ | ??+ | ??+ | ??+ | ??+ | ??+ | ??+ |
| ??9A2 | ??41 | ????2 | ??- | ??- | ??- | ??- | ??- | ??- | ??+ | ??+ | ??+ | ??+ | ??+ |
| ??9A3 | ??32 | ????2 | ??- | ??- | ??- | ??- | ??- | ??- | ??+ | ??+ | ??+ | ??+ | ??+ |
| ??9A4 | ??25 | ????3 | ??- | ??- | ??- | ??- | ??- | ??± | ??+ | ??+ | ??+ | ??+ | ??+ |
| ??9A5 | ??103 | ????2 | ??- | ??- | ??- | ??- | ??- | ??- | ??- | ??+ | ??+ | ??+ | ??+ |
| ??9A6 | ??144 | ????3 | ??- | ??- | ??- | ??- | ??- | ??- | ??- | ??+ | ??+ | ??+ | ??+ |
+ expression positive findings;-expression negative findings; The weak positive findings of ± expression.
Embodiment 5.
Application during different denaturation temperature detection amplified production fragment is polluted.
Adopt the primer among the embodiment 3 that 9A1-9A6 is carried out the polymerase chain reaction, add corresponding amplified fragments in the template and pollute 10ng, 1ng, 100pg, 10pg, 1pg, 10fg, 10fg.With template sample under 95 ℃ and 79 ℃ of two kinds of denaturation temperatures 15 seconds respectively, 62 ℃ (for 9A1,9A2,9A5 and 9A6) or 45 ℃ (for 9A3 and 9A4) annealed and extends totally 25 circulations in 5 seconds.
In the reaction of result's demonstration with 95 ℃ of sex change, no matter whether all primers to having the template pollution all can obtain positive findings, obtain positive findings and only have the template that amplified fragments pollutes under 79 ℃ of denaturation temperatures, do not add and pollute segmental long-chain DNA all be negative (table 6).
Detecting the amplified production fragment under the table 6.79 ℃ denaturation temperature pollutes
| Primer is right | Template is polluted | Amplified production contamination level (wt/20 μ L) | ||||||
| ?10ng | ?1ng | ?100pg | 10pg | ?1pg | ?10fg | ?10fg | ||
| ????9A1 | Do not add | ????- | ??- | ????- | ????- | ??- | ??- | ??- |
| Add | ????+ | ??+ | ????+ | ????+ | ??+ | ??+ | ??+ | |
| ????9A2 | Do not add | ????- | ??- | ????- | ????- | ??- | ??- | ??- |
| Add | ????+ | ??+ | ????+ | ????+ | ??+ | ??+ | ??± | |
| ????9A3 | Do not add | ????- | ??- | ????- | ????- | ??- | ??- | ??- |
| Add | ????+ | ??+ | ????+ | ????+ | ??+ | ??+ | ??+ | |
| ????9A4 | Do not add | ????- | ??- | ????- | ????- | ??- | ??- | ??- |
| Add | ????+ | ??+ | ????+ | ????+ | ??+ | ??+ | ??+ | |
| ????9A5 | Do not add | ????- | ??- | ????- | ????- | ??- | ??- | ??- |
| Add | ????+ | ??+ | ????+ | ????+ | ??+ | ??+ | ??± | |
| ????9A6 | Do not add | ????- | ??- | ????- | ????- | ??- | ??- | ??- |
| Add | ????+ | ??+ | ????+ | ????+ | ??+ | ??+ | ??± | |
+ expression positive findings;-expression negative findings; The weak positive findings of ± expression.
Long-chain DNA can unwind under high-temperature denatured at 95 ℃, and can't sex change under 79 ℃, all can amplify positive findings and pollute fragment 95 ℃ and 79 ℃ of two kinds of denaturation temperatures, utilizes present method
The template that method can significantly be distinguished in the DNA sample to be measured is that the fragment that long-chain DNA or previous amplified production bring is polluted.
Embodiment 6.
Application in the detection of distinguishing genomic dna and cDNA.
Ultrashort product polymerase chain reaction method has its unique application equally in the detection of distinguishing genomic dna and cDNA, the corresponding primer of the ultrashort product of embodiment 3 design is carried out PCR to 9A1-9A6 at genomic dna and cDNA, the template sample is carried out following 2 PCR respectively:
(1) with 95 ℃ of denaturation temperatures 15 seconds, 62 ℃ (for 9A1,9A2,9A5 and 9A6) or 45 ℃ (for 9A3 and 9A4) annealed and extends and carried out initial 2 circulations in 5 seconds; 79 ℃ of sex change of follow-up circulation, 62 ℃ (for 9A1,9A2,9A5 and 9A6) or 45 ℃ (for 9A3 and 9A4) anneals and extended 5 seconds, totally 25 follow-up circulations;
(2) be 1 except that initial circulation, all the other are with (1).
Because genomic dna can unwind at 94-95 ℃, and can't sex change under 68-87 ℃, so genomic dna must have 2 high-temperature denatured circulations at least, just can finish amplification; Only need 1 high-temperature denatured circulation can finish amplification and carry out the complementary DNA (cDNA) that reverse transcription obtains with the single-minded anti-primer of gene.
Actual PCR result shows that genomic dna is positive, and is negative in the reaction (2) in reaction (1); CDNA all is positive in reaction (1) and (2).
Claims (10)
1. the polymerase chain reaction method of a ultrashort product in turn includes the following steps:
(1) template sex change;
(2) primer annealing;
(3) synthetic complementary dna chain is extended in archaeal dna polymerase catalysis down;
(4) set by step (1)-(3) circulation carrying out amplified reaction;
It is characterized in that amplified reaction product length is the 24-150 base.
2. polymerase chain reaction method according to claim 1 is characterized in that amplified reaction product length is the 24-100 base.
3. polymerase chain reaction method according to claim 1 is characterized in that amplified reaction product length is the 40-70 base.
4. according to each described polymerase chain reaction method in the claim 1,2 or 3, it is characterized in that primer length is the 11-40 base.
5. according to each described polymerase chain reaction method in the claim 1,2 or 3, it is characterized in that primer length is the 18-30 base.
6. according to each described polymerase chain reaction method in the claim 1,2 or 3, it is characterized in that reacting initial 2 or 3 circulation denaturation temperatures is 92-96 ℃.
7. according to each described polymerase chain reaction method in the claim 1,2 or 3, it is characterized in that subsequent reactions from the 3rd or the 4th circulation, denaturation temperature is 68-96 ℃.
8. according to each described polymerase chain reaction method in the claim 1,2 or 3, it is characterized in that subsequent reactions from the 3rd or the 4th circulation, denaturation temperature is 72-80 ℃.
9. the application of the described ultrashort product polymerase chain reaction method of claim 1 in detecting the amplified production pollution is characterized in that the template sample carries out initial 2 round-robin reaction with 94-95 ℃ with 68-87 ℃ of two kinds of denaturation temperatures respectively.
10. the application of the described ultrashort product polymerase chain reaction method of claim 1 in the detection of distinguishing genomic dna and cDNA is characterized in that the template sample is carried out 2 PCR respectively:
(1) carries out initial 2 circulations with 94-95 ℃ of denaturation temperature, follow-up circulation 68-87 ℃ sex change;
(2) carry out initial 1 circulation with 94-95 ℃ of denaturation temperature, follow-up circulation 68-87 ℃ sex change.
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA021551847A CN1508258A (en) | 2002-12-18 | 2002-12-18 | Polymerase chain reaction method for expanding ultrashort product and use thereof |
| PCT/CN2003/001063 WO2004055193A1 (en) | 2002-12-18 | 2003-12-15 | Pcr method and application by transnormal low thermo-denaturation temperature |
| AU2003289651A AU2003289651A1 (en) | 2002-12-18 | 2003-12-15 | Pcr method and application by transnormal low thermo-denaturation temperature |
| US11/158,212 US20060063175A1 (en) | 2002-12-18 | 2005-06-15 | Method of polymerase chain reaction with ultra-low denaturing temperatures and applications thereof |
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| CNA021551847A CN1508258A (en) | 2002-12-18 | 2002-12-18 | Polymerase chain reaction method for expanding ultrashort product and use thereof |
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| CN1508258A true CN1508258A (en) | 2004-06-30 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102827828A (en) * | 2011-06-16 | 2012-12-19 | 华东医学生物技术研究所 | Method for eliminating PCR amplification product pollution based on IIs type restriction endonuclease |
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- 2002-12-18 CN CNA021551847A patent/CN1508258A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102827828A (en) * | 2011-06-16 | 2012-12-19 | 华东医学生物技术研究所 | Method for eliminating PCR amplification product pollution based on IIs type restriction endonuclease |
| CN102827828B (en) * | 2011-06-16 | 2014-06-11 | 华东医学生物技术研究所 | Method for eliminating PCR amplification product pollution based on IIs type restriction endonuclease |
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