CN108330151B

CN108330151B - Deoxyribozyme cleavage method

Info

Publication number: CN108330151B
Application number: CN201810131087.4A
Authority: CN
Inventors: 顾宏周
Original assignee: Rudong Bioruler Biotechnology Co ltd
Current assignee: Rudong Bioruler Biotechnology Co ltd
Priority date: 2018-02-09
Filing date: 2018-02-09
Publication date: 2020-06-23
Anticipated expiration: 2038-02-09
Also published as: CN108330151A

Abstract

The invention discloses a thermal cycle assisted deoxyribozyme cleavage method, which is a method for improving the activity (conversion rate of enzyme between substrates) of class I deoxyribozyme capable of cleaving DNA in a trans-hydrolysis manner through a thermal cycle step of heating and cooling, and the method enables k of deoxyribozyme I-R3_catThe value (catalytic constant of the enzyme) is from 0.017min^‑1Increasing to 0.5min^‑1. The reaction mode of thermal cycle can make the deoxyribozyme as a powerful molecular biological tool to efficiently obtain single-stranded DNA fragments.

Description

Deoxyribozyme cleavage method

Technical Field

The invention relates to the fields of biochemistry and molecular biology, in particular to a deoxyribozyme reaction for carrying out trans-cleavage.

Background

For a protease, its catalytic efficiency can be expressed in terms of the turnover number (turn number) of the enzyme. The turnover number of a protease indicates the activity of a catalytic center of an enzyme, and refers to the number of substrate molecules that can be converted per catalytic center (or active center) per unit time (e.g., per second), or the number of moles of substrate that can be converted per unit time per mole of the active center of the enzyme.

The process of cleavage of a specific site on one molecule from a catalytic center on the other molecule by interaction between two nucleic acid molecules is called trans-cleavage. In the trans-cleaved dnazymes, since the enzyme DNA strand and the substrate DNA strand are tightly bound by base complementary pairing, the substrate strand and the enzyme strand are not separated after the reaction, which is a so-called product inhibition effect. In the presence of product inhibition effects, only an equal or excess amount of the DNA strand of the enzyme can be added to cleave the entire substrate.

The construction of DNA nanostructures often requires long, single-stranded DNA, which is not only difficult to achieve by the prior art but also expensive to perform if synthesized by chemical methods, whereas the method of obtaining long, single-stranded DNA by cleaving the genome with dnazymes can reduce the experimental cost by at least 3 orders of magnitude. The existing method for obtaining DNA chains by utilizing deoxyribozymes to cut genomes is only completed through single-conversion cutting reaction, the efficiency is not good, a large amount of DNA chains of enzymes are needed, and the experiment cost is high.

Disclosure of Invention

Aiming at the technical problems and based on Zn²⁺Depending on the research of the reaction rate and the optimal reaction temperature of the class I deoxyribozyme, the invention provides a thermal cycling process which assists one deoxyribozyme to cut a plurality of substrate sequences, namely, increases the conversion rate of the deoxyribozyme, thereby improving the reaction rate.

The invention firstly provides a deoxyribozyme cleavage method, which comprises the following steps:

1) heating a class I deoxyribozyme reaction system to a chain denaturation temperature, and keeping for a period of time to ensure the full denaturation of a substrate chain and a polymerase chain;

2) then the temperature is reduced to the renaturation and cutting temperature, so that the renaturation and cutting reaction can be carried out, and the temperature is kept for a period of time.

Further, steps 1) -2) are repeated until most of the substrate is cleaved.

Preferably, in the above step, the substrate chain in the reaction system of the class I deoxyribozyme is in excess.

Further, the denaturation temperature in step 1) is preferably 65 to 95 ℃, more preferably 65 to 80 ℃, and most preferably 70 ± 2 ℃.

Further, the time for denaturation in step 1) is at least 15 seconds, preferably 20 seconds or more, more preferably 20 to 30 seconds, and most preferably 20 to 25 seconds.

Further, the renaturation and cleavage temperature in step 2) is preferably 37 to 55 ℃, most preferably 45 ± 2 ℃.

Further, the time for renaturation and cleavage in step 2) is at least 30 seconds, preferably 1 minute or more, more preferably 1 to 5 minutes, and most preferably 1 to 2 minutes.

Preferably, the substrate is the M13mp18 genome.

Preferably, the class I deoxyribozyme is I-R3. I-R3 is a thermophilic deoxyribozyme, and the cutting rate of the deoxyribozyme is gradually increased when the temperature is increased from 37 ℃ to 54 ℃ on the premise of ensuring the stability of the secondary structure. There are two stem structures of the deoxyribozyme I-R3, each containing 16 bases and 14 bases, as shown in FIG. 1. About more than 90% of the substrate sequence was completely cleaved by holding at 45 ℃ for 1 minute. However, at 60 ℃ cleavage hardly occurs because the high temperature destroys the secondary structure of the deoxyribozyme.

Preferably, the group I deoxyribozyme is one or more of E1, E2, E3 and E4, wherein E1 has a sequence shown as SEQ NO.1, E2 has a sequence shown as SEQ NO.2, E3 has a sequence shown as SEQ NO.3, and E4 has a sequence shown as SEQ NO. 4.

E1(SEQ NO.1)：

5′-AACCAGACCGGAAGCAAATAGTTGAGCTAGGTCAGGATTAGAGAGT-3′；

E2(SEQ NO.2)：

5′-TTTCGTCACCAGTACAAATAGTTGAGCTGCCTGTAGCATTCCACAG-3′；

E3(SEQ NO.3)：

5′-ACTATTAAAGAACGTGGATAGTTGAGCTGTCAAAGGGCGAAAAACC-3′；

E4(SEQ NO.4)：

5′-GGTCAGTATTAACACCGCTAGTTGAGCTAGTGCCACGCTGAGAGCC-3′。

Further, the class I deoxyribozyme has a 10-base sequence of I-R3 enzyme domain, i.e., 5'-TAGTTGAGCT-3' (SEQ NO. 5).

The invention further provides a thermal cycle assisted cleavage method of deoxyribozyme I-R3, which comprises the following steps: in the case of excess substrate chain, the I-R3 reaction was heated to 70. + -. 1 ℃ for at least 20 seconds to ensure adequate denaturation; then the temperature is reduced to 45 +/-1 ℃ to ensure that the renaturation and the cutting reaction are carried out, and the temperature is kept for at least 1 minute to ensure that the cutting reaction is completely carried out; raising the temperature to 70 +/-1 ℃ again, and keeping the temperature for at least 20 seconds to denature and separate the enzyme chain and the cut substrate chain; then cooling to 45 +/-1 ℃ again, keeping for at least 1 minute, combining the substrate chain which is not cut with the enzyme chain, and carrying out a new round of cutting reaction; this is done for a number of rounds until most of the substrate has been cleaved. The reaction scheme of the above steps is shown in FIG. 2.

In a preferred embodiment of the present invention, in the I-R3 reaction system, the enzyme and the substrate are mixed at a molar ratio of 1:50, and each cycle is carried out for 1 minute and 20 seconds, and it takes 120 minutes to carry out 90 cycles, which is equivalent to every cycleIt takes 2 minutes 40 seconds for a transition to occur, then k corresponds to_catThe value was 0.375min^-1. If 60 cycles are performed, 80% of the substrate will be cleaved, k_catThe value is 0.5min^-1The conversion rate is 30 times higher than that of the previously reported deoxyribozyme. In another preferred embodiment of the present invention, the same conversion rate (0.3-0.5 min) can be obtained by mixing the enzyme and the substrate in a molar ratio of 1:200 in the reaction system of I-R3^-1). As shown in fig. 3 and 4.

The invention also provides a method for efficiently obtaining the M13mp18 genome DNA fragment by using the deoxyribozyme cutting method. Wherein the full-length sequence of the M13mp18 genome DNA is shown as SEQ NO. 6.

The M13 phage is a filamentous phage with a circular single-stranded DNA molecule inside. Due to the variability of base 13 of the catalytic core region of the deoxyribozyme I-R3 (fig. 1), and the presence of only 6 highly conserved sites in the substrate sequence, this resulted in the discovery of 4 potential cleavage sites (S1, S2, S3 and S4) of class I deoxyribozymes in the M13mp18 genome (fig. 5). Wherein S1, S2 and S3 are found to be good substrate sequences of class I deoxyribozyme I-R3, the S4 contains a mutation site (R13C), the base A at the R13 position of the deoxyribozyme I-R3 is mutated into the base C at 37 ℃, and the reaction rate is 0.067min^-1The reduction is about 15 times compared with the wild type. The cleavage signal was 80% only after 20 min at 37 ℃. Therefore, the mutant site (S4) contained in the M13 genome had a very low reaction efficiency when the DNAzyme E4 was added under conventional conditions (37 ℃ C.). However, if the thermal cycle-assisted method is used to cut the M13 genome, the reaction temperature can be increased to 50 ℃ to further accelerate the cutting speed. In addition, the reaction time was extended from the original 1 minute to 2 or 5 minutes to ensure that almost all of the substrate sequence was bound and cleaved by the enzyme sequence.

Therefore, the method for efficiently obtaining the M13mp18 genomic DNA fragment provided by the invention comprises the following steps:

mixing an excessive M13mp18 genome with at least one type I deoxyribozyme, and preparing a thermal cycling reaction system; multiple thermal cycles are then performed, each cycle at 70 ± 1 ℃ for at least 20 seconds, 50 ± 1 ℃ for more than 1 minute, or more preferably more than 2 minutes, until most of the substrate is cleaved.

Preferably, in the method for efficiently obtaining the M13mp18 genome DNA fragment, the class I deoxyribozyme is one or more of E1, E2, E3 and E4, and is further preferably a mixture of E1, E2, E3 and E4.

Wherein, when the type I deoxyribozyme is E1 or/and E3, the temperature is kept for more than 2 minutes at 50 +/-1 ℃, and when the type I deoxyribozyme is E2 or/and E4, the temperature is kept for more than 5 minutes at 50 +/-1 ℃.

Preferably, the mixed molar ratio of the class I deoxyribozyme to the M13mp18 genome is 1: 50.

Preferably, the number of thermal cycles is 90 or more, and most preferably 100.

In a preferred embodiment of the present invention, four deoxyribozymes (E1-4) were aligned and combined and mixed with M13mp18 genome at a molar ratio of 1:50, respectively, and then subjected to 100 thermal cycles of 70 ℃ for 20 seconds and 50 ℃ for 2 or 5 minutes, and the size of the cleaved DNA fragment was observed by 0.8% denatured agar gel. For the reaction of cutting M13 genome by multi-conversion deoxyribozyme, 100 cycles are carried out, 40 conversions can be carried out, and the cutting percentage is more than 80%. Compared to many proteases, despite their reaction turnover (. about.0.07-0.17 min)^-1) It is also slightly inferior, but this thermal cycling-assisted method of increasing the turnover rate of deoxyribozymes has resulted in nearly two orders of magnitude improvement in the turnover rate of deoxyribozymes upon cleavage than previously reported.

The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.

Drawings

FIG. 1 is a schematic diagram of the secondary structure of deoxyribozyme I-R3;

FIG. 2 is a schematic diagram of an enzyme digestion reaction with thermal cycling;

FIG. 3 is a graph showing the results of electrophoresis at different ratios of enzyme and substrate, and at different numbers of cycles of reaction;

FIG. 4 is a graph of the results of the percentage of cleavage after reaction for different numbers of cycles, with different ratios of enzyme to substrate being counted;

FIG. 5 is a schematic diagram of the deoxyribozyme cleavage site (S1-4) and S4 sequence on M13mp 18;

FIG. 6 shows the results of M13mp18 agarose electrophoresis after cleavage with different DNAzymes.

Detailed Description

Example 1 measurement of catalytic constant of a thermal cycling-assisted deoxyribozyme

① reaction System 5pmol of 5' end was subjected to³²P-labeled DNA substrate strand (I-R3S) and 0.1pmol or 0.025pmol of DNA polymerase chain (I-R3E) were mixed in a molar ratio of enzyme to substrate of 1:50 or 1:200, respectively, in a reaction system of 200. mu.L.

The sequence of I-R3S is:

5′-CTCGTGATGCAGACGTTGAAGGATTATCTTTCTGACT-3′(SEQ NO.7)；

the sequence of I-R3E is:

5′-AGTCAGAAAGATAATCTAGTTGAGCTGTCTGCATCACGAG-3′(SEQ NO.8)；

reaction buffer:

② reaction conditions 70 ℃ for 20 seconds and 45 ℃ for 1 minute.

③ detection of products 5. mu.L of a sample was added to the stop buffer for every 5 cycles of the reaction in which the enzyme and the substrate were mixed at a molar ratio of 1:50, 5. mu.L of a sample was added to the stop buffer for every 20 cycles of the reaction in which the enzyme and the substrate were mixed at a molar ratio of 1: 200. samples collected at different reaction cycles were analyzed by 10% denaturing polyacrylamide gel electrophoresis, and the results are shown in FIGS. 3 and 4.

Example 2 thermal cycle assisted DNAzyme cleavage M13mp18 genome experiment

① reaction system, mixing enzyme (E1, E2, E3, E4, E1+ E2, E1+ E3, E1+ E4) and substrate M13DNA according to a molar ratio of 1:50 (the ratio of the mixed enzyme to the substrate is 1:1:50), namely:

the amount of M13DNA used was: 1 μ g (. about.0.4 pmol)

The dosage of the deoxyribozymes E1, E2, E3 and E4 is as follows: about 0.008pmol

② reaction conditions 100 cycles of

Temperature and time settings for each cycle:

e1, E3, E1+ E3: at 70 ℃ for 20 seconds and at 50 ℃ for 2 minutes

E2, E4, E1+ E2, E1+ E4: at 70 ℃ for 20 seconds and at 50 ℃ for 5 minutes

③ detection of the product was analyzed by electrophoresis on 0.8% denatured agar gel containing 2M formaldehyde, the results are shown in FIG. 6.

It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.

Sequence listing

<110> Bai ao Baile Biotech Co., Ltd, Dongbao Baile

<120> a deoxyribozyme cleavage method

<130>BABL445-181002A

<141>2018-02-07

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<170>SIPOSequenceListing 1.0

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aaccagaccg gaagcaaata gttgagctag gtcaggatta gagagt 46

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tttcgtcacc agtacaaata gttgagctgc ctgtagcatt ccacag 46

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actattaaag aacgtggata gttgagctgt caaagggcga aaaacc 46

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ggtcagtatt aacaccgcta gttgagctag tgccacgctg agagcc 46

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<212>DNA

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tagttgagct 10

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<211>7249

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<213>phage M13

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aatgctacta ctattagtag aattgatgcc accttttcag ctcgcgcccc aaatgaaaat 60

atagctaaac aggttattga ccatttgcga aatgtatcta atggtcaaac taaatctact 120

cgttcgcaga attgggaatc aactgttata tggaatgaaa cttccagaca ccgtacttta 180

gttgcatatt taaaacatgt tgagctacag cattatattc agcaattaag ctctaagcca 240

tccgcaaaaa tgacctctta tcaaaaggag caattaaagg tactctctaa tcctgacctg 300

ttggagtttg cttccggtct ggttcgcttt gaagctcgaa ttaaaacgcg atatttgaag 360

tctttcgggc ttcctcttaa tctttttgat gcaatccgct ttgcttctga ctataatagt 420

cagggtaaag acctgatttt tgatttatgg tcattctcgt tttctgaact gtttaaagca 480

tttgaggggg attcaatgaa tatttatgac gattccgcag tattggacgc tatccagtct 540

aaacatttta ctattacccc ctctggcaaa acttcttttg caaaagcctc tcgctatttt 600

ggtttttatc gtcgtctggt aaacgagggt tatgatagtg ttgctcttac tatgcctcgt 660

aattcctttt ggcgttatgt atctgcatta gttgaatgtg gtattcctaa atctcaactg 720

atgaatcttt ctacctgtaa taatgttgtt ccgttagttc gttttattaa cgtagatttt 780

tcttcccaac gtcctgactg gtataatgag ccagttctta aaatcgcata aggtaattca 840

caatgattaa agttgaaatt aaaccatctc aagcccaatt tactactcgt tctggtgttt 900

ctcgtcaggg caagccttat tcactgaatg agcagctttg ttacgttgat ttgggtaatg 960

aatatccggt tcttgtcaag attactcttg atgaaggtca gccagcctat gcgcctggtc 1020

tgtacaccgt tcatctgtcc tctttcaaag ttggtcagtt cggttccctt atgattgacc 1080

gtctgcgcct cgttccggct aagtaacatg gagcaggtcg cggatttcga cacaatttat 1140

caggcgatga tacaaatctc cgttgtactt tgtttcgcgc ttggtataat cgctgggggt 1200

caaagatgag tgttttagtg tattcttttg cctctttcgt tttaggttgg tgccttcgta 1260

gtggcattac gtattttacc cgtttaatgg aaacttcctc atgaaaaagt ctttagtcct 1320

caaagcctct gtagccgttg ctaccctcgt tccgatgctg tctttcgctg ctgagggtga 1380

cgatcccgca aaagcggcct ttaactccct gcaagcctca gcgaccgaat atatcggtta 1440

tgcgtgggcg atggttgttg tcattgtcgg cgcaactatc ggtatcaagc tgtttaagaa 1500

attcacctcg aaagcaagct gataaaccga tacaattaaa ggctcctttt ggagcctttt 1560

ttttggagat tttcaacgtg aaaaaattat tattcgcaat tcctttagtt gttcctttct 1620

attctcactc cgctgaaact gttgaaagtt gtttagcaaa atcccataca gaaaattcat 1680

ttactaacgt ctggaaagac gacaaaactt tagatcgtta cgctaactat gagggctgtc 1740

tgtggaatgc tacaggcgtt gtagtttgta ctggtgacga aactcagtgt tacggtacat 1800

gggttcctat tgggcttgct atccctgaaa atgagggtgg tggctctgag ggtggcggtt 1860

ctgagggtgg cggttctgag ggtggcggta ctaaacctcc tgagtacggt gatacaccta 1920

ttccgggcta tacttatatc aaccctctcg acggcactta tccgcctggt actgagcaaa 1980

accccgctaa tcctaatcct tctcttgagg agtctcagcc tcttaatact ttcatgtttc 2040

agaataatag gttccgaaat aggcaggggg cattaactgt ttatacgggc actgttactc 2100

aaggcactga ccccgttaaa acttattacc agtacactcc tgtatcatca aaagccatgt 2160

atgacgctta ctggaacggt aaattcagag actgcgcttt ccattctggc tttaatgagg 2220

atttatttgt ttgtgaatat caaggccaat cgtctgacct gcctcaacct cctgtcaatg 2280

ctggcggcgg ctctggtggt ggttctggtg gcggctctga gggtggtggc tctgagggtg 2340

gcggttctga gggtggcggc tctgagggag gcggttccgg tggtggctct ggttccggtg 2400

attttgatta tgaaaagatg gcaaacgcta ataagggggc tatgaccgaa aatgccgatg 2460

aaaacgcgct acagtctgac gctaaaggca aacttgattc tgtcgctact gattacggtg 2520

ctgctatcga tggtttcatt ggtgacgttt ccggccttgc taatggtaat ggtgctactg 2580

gtgattttgc tggctctaat tcccaaatgg ctcaagtcgg tgacggtgat aattcacctt 2640

taatgaataa tttccgtcaa tatttacctt ccctccctca atcggttgaa tgtcgccctt 2700

ttgtctttgg cgctggtaaa ccatatgaat tttctattga ttgtgacaaa ataaacttat 2760

tccgtggtgt ctttgcgttt cttttatatg ttgccacctt tatgtatgta ttttctacgt 2820

ttgctaacat actgcgtaat aaggagtctt aatcatgcca gttcttttgg gtattccgtt 2880

attattgcgt ttcctcggtt tccttctggt aactttgttc ggctatctgc ttacttttct 2940

taaaaagggc ttcggtaaga tagctattgc tatttcattg tttcttgctc ttattattgg 3000

gcttaactca attcttgtgg gttatctctc tgatattagc gctcaattac cctctgactt 3060

tgttcagggt gttcagttaa ttctcccgtc taatgcgctt ccctgttttt atgttattct 3120

ctctgtaaag gctgctattt tcatttttga cgttaaacaa aaaatcgttt cttatttgga 3180

ttgggataaa taatatggct gtttattttg taactggcaa attaggctct ggaaagacgc 3240

tcgttagcgt tggtaagatt caggataaaa ttgtagctgg gtgcaaaata gcaactaatc 3300

ttgatttaag gcttcaaaac ctcccgcaag tcgggaggtt cgctaaaacg cctcgcgttc 3360

ttagaatacc ggataagcct tctatatctg atttgcttgc tattgggcgc ggtaatgatt 3420

cctacgatga aaataaaaac ggcttgcttg ttctcgatga gtgcggtact tggtttaata 3480

cccgttcttg gaatgataag gaaagacagc cgattattga ttggtttcta catgctcgta 3540

aattaggatg ggatattatt tttcttgttc aggacttatc tattgttgat aaacaggcgc 3600

gttctgcatt agctgaacat gttgtttatt gtcgtcgtct ggacagaatt actttacctt 3660

ttgtcggtac tttatattct cttattactg gctcgaaaat gcctctgcct aaattacatg 3720

ttggcgttgt taaatatggc gattctcaat taagccctac tgttgagcgt tggctttata 3780

ctggtaagaa tttgtataac gcatatgata ctaaacaggc tttttctagt aattatgatt 3840

ccggtgttta ttcttattta acgccttatt tatcacacgg tcggtatttc aaaccattaa 3900

atttaggtca gaagatgaaa ttaactaaaa tatatttgaa aaagttttct cgcgttcttt 3960

gtcttgcgat tggatttgca tcagcattta catatagtta tataacccaa cctaagccgg 4020

aggttaaaaa ggtagtctct cagacctatg attttgataa attcactatt gactcttctc 4080

agcgtcttaa tctaagctat cgctatgttt tcaaggattc taagggaaaa ttaattaata 4140

gcgacgattt acagaagcaa ggttattcac tcacatatat tgatttatgt actgtttcca 4200

ttaaaaaagg taattcaaat gaaattgtta aatgtaatta attttgtttt cttgatgttt 4260

gtttcatcat cttcttttgc tcaggtaatt gaaatgaata attcgcctct gcgcgatttt 4320

gtaacttggt attcaaagca atcaggcgaa tccgttattg tttctcccga tgtaaaaggt 4380

actgttactg tatattcatc tgacgttaaa cctgaaaatc tacgcaattt ctttatttct 4440

gttttacgtg caaataattt tgatatggta ggttctaacc cttccattat tcagaagtat 4500

aatccaaaca atcaggatta tattgatgaa ttgccatcat ctgataatca ggaatatgat 4560

gataattccg ctccttctgg tggtttcttt gttccgcaaa atgataatgt tactcaaact 4620

tttaaaatta ataacgttcg ggcaaaggat ttaatacgag ttgtcgaatt gtttgtaaag 4680

tctaatactt ctaaatcctc aaatgtatta tctattgacg gctctaatct attagttgtt 4740

agtgctccta aagatatttt agataacctt cctcaattcc tttcaactgt tgatttgcca 4800

actgaccaga tattgattga gggtttgata tttgaggttc agcaaggtga tgctttagat 4860

ttttcatttg ctgctggctc tcagcgtggc actgttgcag gcggtgttaa tactgaccgc 4920

ctcacctctg ttttatcttc tgctggtggt tcgttcggta tttttaatgg cgatgtttta 4980

gggctatcag ttcgcgcatt aaagactaat agccattcaa aaatattgtc tgtgccacgt 5040

attcttacgc tttcaggtca gaagggttct atctctgttg gccagaatgt cccttttatt 5100

actggtcgtg tgactggtga atctgccaat gtaaataatc catttcagac gattgagcgt 5160

caaaatgtag gtatttccat gagcgttttt cctgttgcaa tggctggcgg taatattgtt 5220

ctggatatta ccagcaaggc cgatagtttg agttcttcta ctcaggcaag tgatgttatt 5280

actaatcaaa gaagtattgc tacaacggtt aatttgcgtg atggacagac tcttttactc 5340

ggtggcctca ctgattataa aaacacttct caggattctg gcgtaccgtt cctgtctaaa 5400

atccctttaa tcggcctcct gtttagctcc cgctctgatt ctaacgagga aagcacgtta 5460

tacgtgctcg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg 5520

tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt 5580

cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg 5640

ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga 5700

tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac 5760

gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc 5820

tatctcgggc tattcttttg atttataagg gattttgccg atttcggaac caccatcaaa 5880

caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc 5940

caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg 6000

gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 6060

cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct 6120

cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat 6180

tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg aattcgagct 6240

cggtacccgg ggatcctcta gagtcgacct gcaggcatgc aagcttggca ctggccgtcg 6300

ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac 6360

atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac 6420

agttgcgcag cctgaatggc gaatggcgct ttgcctggtt tccggcacca gaagcggtgc 6480

cggaaagctg gctggagtgc gatcttcctg aggccgatac tgtcgtcgtc ccctcaaact 6540

ggcagatgca cggttacgat gcgcccatct acaccaacgt gacctatccc attacggtca 6600

atccgccgtt tgttcccacg gagaatccga cgggttgtta ctcgctcaca tttaatgttg 6660

atgaaagctg gctacaggaa ggccagacgc gaattatttt tgatggcgtt cctattggtt 6720

aaaaaatgag ctgatttaac aaaaatttaa tgcgaatttt aacaaaatat taacgtttac 6780

aatttaaata tttgcttata caatcttcct gtttttgggg cttttctgat tatcaaccgg 6840

ggtacatatg attgacatgc tagttttacg attaccgttc atcgattctc ttgtttgctc 6900

cagactctca ggcaatgacc tgatagcctt tgtagatctc tcaaaaatag ctaccctctc 6960

cggcattaat ttatcagcta gaacggttga atatcatatt gatggtgatt tgactgtctc 7020

cggcctttct cacccttttg aatctttacc tacacattac tcaggcattg catttaaaat 7080

atatgagggt tctaaaaatt tttatccttg cgttgaaata aaggcttctc ccgcaaaagt 7140

attacagggt cataatgttt ttggtacaac cgatttagct ttatgctctg aggctttatt 7200

gcttaatttt gctaattctt tgccttgcct gtatgattta ttggatgtt 7249

<210>7

<211>37

<212>DNA

<213>Artificial Sequence

<400>7

ctcgtgatgc agacgttgaa ggattatctt tctgact 37

<210>8

<211>40

<212>DNA

<213>Artificial Sequence

<400>8

agtcagaaag ataatctagt tgagctgtct gcatcacgag 40

Claims

1. A deoxyribozyme cleavage method, characterized by comprising the steps of:

1) heating a class I deoxyribozyme reaction system containing class I deoxyribozyme to a chain denaturation temperature, wherein the denaturation temperature is 70 +/-1 ℃, and keeping the temperature for at least 20 seconds to ensure the full denaturation of a substrate chain and a polymerase chain; the gene sequence of the class I deoxyribozyme is shown as SEQ ID NO.8, and the substrate chain in the class I deoxyribozyme reaction system is excessive; the substrate chain is M13mp18 genome;

2) then the temperature is reduced to 50 +/-1 ℃, so that the substrate chain and the enzyme chain are subjected to renaturation and cutting reaction, the temperature is maintained for at least 1 minute, and the cutting reaction is completely carried out;

3) repeating the steps 1) and 2) for more than 90 times.