CN105400771B - A kind of method for obtaining known array flank unknown nucleotide sequence - Google Patents
A kind of method for obtaining known array flank unknown nucleotide sequence Download PDFInfo
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- CN105400771B CN105400771B CN201510910524.9A CN201510910524A CN105400771B CN 105400771 B CN105400771 B CN 105400771B CN 201510910524 A CN201510910524 A CN 201510910524A CN 105400771 B CN105400771 B CN 105400771B
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Abstract
The invention discloses a kind of methods for obtaining known array flank unknown nucleotide sequence, include the following steps：1）According to known two specific primers of sequence design, labeled as SP1 and SP2, SP2 band restriction enzyme enzyme recognition sites, in SP2 to a pair of of inverse PCR primer F1 and D1 of design between border sequence；2）Using genomic DNA be template with any one degenerate primer and special SP1 primers, amplification obtains PCR product 1；3）It is template with the PCR product 1, uses special SP2 primers and step 2）In it is of the same race but add in restriction enzyme enzyme recognition site degenerate primer expand to obtain PCR product 2；4）The cyclisation of T4 ligases will be added in after 2 digestion of PCR product, as template, PCR product 3 is obtained using F1 and D1 primer amplifications, so as to obtain the flanking sequence.The method provided by the present invention has the following advantages：Operate that simple, of low cost, success rate is high, it is short etc. to take.
Description
Technical field
The present invention relates to genome unknown nucleotide sequences to expand field, and in particular to a kind of acquisition known array flank unknown nucleotide sequence
Method.
Background technology
Flank amplification is the key technology for verifying gene order.The method of traditional amplification unknown nucleotide sequence is segmented into two
Kind：One kind is to be cyclized sequence, and design reverse primer amplification unknown nucleotide sequence has species specificity, effectively for the enzyme of digestion
The probability of amplification is low, influences success rate；Another kind is using specific primer and degenerate primer amplification unknown nucleotide sequence, this kind of method
Non-specific amplification is more, influences success rate.The comprehensive two methods of the present invention first introduce degenerate primer amplification flanking sequence, logical
It is expanded after crossing cohesive end cyclisation product, amplification efficiency is improved while reducing non-specific amplification, increase success rate.
Invention content
The purpose of the present invention is to provide a kind of methods for obtaining known array flank unknown nucleotide sequence, can be with using this method
Unknown flanking sequence is obtained, easy to operate, of low cost, success rate is high, takes short.
To achieve the above object, the present invention adopts the following technical scheme that：
The method of the present invention is specific as follows：
A kind of method for obtaining known array flank unknown nucleotide sequence, includes the following steps：
(1) known array, the consistent specific primer in two directions of design, from separate border sequence to by proximal border are selected
The sequence of sequence successively labeled as SP1 and SP2, SP2 band restriction enzyme enzyme recognition site, SP2 between border sequence again
A pair of of inverse PCR primer F1 and D1 of design；
(2) using genomic DNA as template, first round PCR amplification is carried out with the SP1 respectively with 6 kinds of degenerate primer AD, is obtained
To the PCR product 1 for having amplified band；
(3) there is the PCR product 1 of amplified band for template with described, using same in special SP2 primers and step (2)
The corresponding degenerate primer AD ' amplifications of kind but addition restriction enzyme enzyme recognition site obtain PCR product 2；
(4) cyclisation of T4 ligases will be added in after 2 digestion of PCR product, as template, the use of F1 and D1 is primer, expands
PCR product 3 is obtained, flanking sequence is obtained from the PCR product 3.
The degenerate primer AD and degenerate primer AD ' for adding in restriction enzyme enzyme recognition site is as follows：
AD1：NTCGASTWTSGWGTT、AD’1：NTCGASTWTSGWGTTCCCGGG ；
AD2：NGTCGASWGANAWGAA、 AD’2 ：NGTCGASWGANAWGAACCCGGG；
AD3：NGTASASWGTNAWCAA、 AD’3：NGTASASWGTNAWCAACCCGGG；
AD4：STTGNTASTNCTNTGC、AD’4：STTGNTASTNCTNTGCCCCGGG ；
AD5：AGWGNAGWANCAWAGG、 AD’5：AGWGNAGWANCAWAGGCCCGGG；
AD6 ：TGWGNAGWANCASAGA、 AD’6：TGWGNAGWANCASAGACCCGGG.
Advantage body of the present invention is：
This method, which is effectively reduced, introduces the non-specific amplification that brings of degenerate primer, operation is simple, of low cost, into
Power is high, it is short to take.
Description of the drawings
Fig. 1 is a kind of flow chart wherein a for the method for obtaining known array flank unknown nucleotide sequence：It is set on known array
Two specific primer SP1, SP2 in the same direction are counted, SP2 band restriction enzyme enzyme recognition sites design between SP2 and border sequence
A pair of of reverse primer F1, D1.b：SP1 and AD is primer, and genomic DNA is template amplification.c：Use SP2 and AD ' it is primer, it walks
Rapid b products are template amplification.d：The amplified production that step c is obtained.e：The production obtained using digestion with restriction enzyme step c
Object.f：Using the product in ligase cyclisation step e, and the use of F1, D1 is primer amplification as template.g：It obtains
Know sequence flank unknown nucleotide sequence segment.
Fig. 2 is illustrated for design of primers, and two specific primer SP1, SP2 in the same direction, SP2 band limitations are designed on known array
Property endonuclease recognized site, a pair of of reverse primer F1, D1 are designed between SP1 and border sequence.
Fig. 3 is asymmetric primer first time, second of amplification electrophoretogram, and 1 is asymmetric primer first time amplified production electricity
Swimming band, 2 be asymmetric second of amplified production electrophoretic band of primer.
Fig. 4 is that digestion products detect and using cyclisation product as template PCR amplifications unknown nucleotide sequence product electrophoretogram, and 1 is digestion
Product detection electrophoretic band, 2 be using cyclisation product as template PCR amplifications unknown nucleotide sequence product electrophoretogram.
Specific embodiment
Embodiment 1
1st, DNA extractions and purifying：
Various general DNA extraction methods and purification process can be used, extract the genome DNA purified.
2nd, design of primers：
The sequence of roxburgh anoectochilus terminal bud PAL gene intermediate segments that is obtained by homogenetic amplification is selected, it is consistent to design two directions
Specific primer, the sequence from far from border sequence to close to border sequence are labeled as 5 '-TGGTGTCACTACTGG of SP1 successively
ATTTGG-3 ' and SP2 5 '-CCCGGGGCATCCGATTCGAAATCCT-3 ', SP2 with restriction enzyme enzyme recognition site 5 '-
CCCGGG-3 ', in SP2 to a pair of of inverse PCR primer F1 of redesign between border sequence：5’-CAAGATCGCCGGAGGCGGTG-
3 ' and D1：5 '-CTTACATTGCGGGTGTCTTAACC-3 ', such as Fig. 2.
3rd, unknown nucleotide sequence expands：
A, asymmetric primer expands for the first time：
Using genomic DNA as template, the expansion of first round PCR is carried out with the SP1 respectively with 6 kinds of different degenerate primer AD
Increase, amplification system is：
TaqPlusPCR Master Mix 20ul,
DNA profiling 2ul,
2* primers（AD and SP1）2ul,
ddH2O 16ul；
Amplification program is：
94 DEG C, 30min；
94 DEG C, 30s, 68 DEG C, 1min；72 DEG C, 2.5min（5 cycles）；
94 DEG C, 30s；25 DEG C, 3min, ramping to 72 DEG C, 3 min of over；72 DEG C, 2.5min；
94 DEG C, 15s；68 DEG C, 1min；72 DEG C, 2.5min；94 DEG C, 15s；68 DEG C, 1min；72 DEG C, 2.5min；94 DEG C,
15s；44 DEG C, 1min；72 DEG C, 2.5min（15 cycles）；
72 DEG C, 8min；12 DEG C of heat preservations.
B, asymmetric second of amplification of primer：
There is amplified band using degenerate primer AD3 in upper step A, using this product as template, using with restriction enzyme
Degenerate primer AD ' the 35 '-NGTASASWGTNAWCAACCCGGG-3 ' and the SP2 of enzyme recognition site carry out the expansion of first round PCR
Increase, amplification system is：
TaqPlusPCR Master Mix 20ul,
DNA profiling 2ul,
2* primers（AD ' and SP2）2ul,
ddH2O 16ul；
Amplification program is：
94 DEG C, 30min；
94 DEG C, 15s；68 DEG C, 1min；72 DEG C, 2.5min；94 DEG C, 15s；68 DEG C, 1min；72 DEG C, 2.5min；94 DEG C,
15s；44 DEG C, 1min；72 DEG C, 2.5min（12 cycles）；
72 DEG C, 8min；12 DEG C of heat preservations.
C, product is cyclized：
10u Xma I digestions are added in the product that upper step B is expanded, with molten after two volumes ethanol precipitation after digestion
In 20ul ET, 1u T4 ligases are added, 16 DEG C preserve overnight, place into 65 DEG C of preservation 10min, inactivate ligase, obtain
To cyclisation product.
D, PCR amplification
It is template after the product recycling that above step C is cyclized, using F1 and D1 as primer, regular-PCR program expands
To product be unknown flanking fragment.Amplification system is：
TaqPlusPCR Master Mix 20ul,
DNA profiling 2ul,
2* primers（F1 and D1）2ul,
ddH2O 16ul。
4th, the sequencing of sequence compares
The unknown flanking fragment obtained using 2% non denatured agarose gel electrophoresis detection amplification, by segment after glue recycling
PMD18-T carriers are connected to, PMD18-T universal primer detection primer sequences are used, and carry out sequence ratio after detecting positive bacterium solution
It is right.
1 degenerate primer sequence of table
Annotation：S represents G/C, and W represents A/T, and N represents A/T/C/G.
Interpretation of result：
Amplification electrophoretic band shows non-specific amplification to asymmetric primer for the first time in Fig. 3, and specific amplification and non-
Specific amplification band cannot be distinguished from.Second of amplification electrophoretic band shows that non-specific amplification significantly reduces, but because introduces
Degenerate primer, however it remains non-specific amplification.Digestion products test strip is weaker in Fig. 4.Using cyclisation product as template PCR
Amplification unknown nucleotide sequence product electrophoretogram shows that electrophoretic band is single, and band is clear, and expanding effect is preferable.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification should all belong to the covering scope of the present invention.
SEQUENCE LISTING
<110>Sanming College
<120>A kind of method for obtaining known array flank unknown nucleotide sequence
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<170> PatentIn version 3.3
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ngtasaswgt nawcaacccg gg 22
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<220>
<221> misc_feature
<222> (13)..(13)
<223> n is a, c, g, or t
<400> 7
sttgntastn ctntgc 16
<210> 8
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (5)..(5)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (10)..(10)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (13)..(13)
<223> n is a, c, g, or t
<400> 8
sttgntastn ctntgccccg gg 22
<210> 9
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<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (5)..(5)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (10)..(10)
<223> n is a, c, g, or t
<400> 9
agwgnagwan cawagg 16
<210> 10
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (5)..(5)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (10)..(10)
<223> n is a, c, g, or t
<400> 10
agwgnagwan cawaggcccg gg 22
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<220>
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<222> (10)..(10)
<223> n is a, c, g, or t
<400> 11
tgwgnagwan casaga 16
<210> 12
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (5)..(5)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (10)..(10)
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<400> 12
tgwgnagwan casagacccg gg 22
<210> 13
<211> 21
<212> DNA
<213>Artificial sequence
<400> 13
tggtgtcact actggatttg g 21
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cccggggcat ccgattcgaa atcct 25
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<211> 6
<212> DNA
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cccggg 6
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<211> 20
<212> DNA
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<400> 16
caagatcgcc ggaggcggtg 20
<210> 17
<211> 23
<212> DNA
<213>Artificial sequence
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cttacattgc gggtgtctta acc 23
Claims (2)
- A kind of 1. method for obtaining known array flank unknown nucleotide sequence, it is characterised in that：Include the following steps：(1) known array, the consistent specific primer in two directions of design, from far from border sequence to close border sequence are selected Sequence successively labeled as SP1 and SP2, SP2 band restriction enzyme enzyme recognition site, redesigned in SP2 between border sequence A pair of of inverse PCR primer F1 and D1；(2) using genomic DNA as template, first round PCR amplification is carried out with the SP1 respectively with 6 kinds of degenerate primer AD, is had The PCR product 1 of amplified band；(3) have the PCR product 1 of amplified band for template with described, using of the same race in special SP2 primers and step (2) but The corresponding degenerate primer AD ' amplifications for adding in restriction enzyme enzyme recognition site obtain PCR product 2；(4) cyclisation of T4 ligases will be added in after 2 digestion of PCR product, the use of F1 and D1 is primer as template, amplification obtains PCR product 3 obtains flanking sequence from the PCR product 3.
- 2. a kind of method for obtaining known array flank unknown nucleotide sequence according to claim 1, it is characterised in that：It is described simultaneous And the degenerate primer AD ' of primer AD and addition restriction enzyme enzyme recognition site is as follows：AD1：NTCGASTWTSGWGTT、AD’1：NTCGASTWTSGWGTTCCCGGG ；AD2：NGTCGASWGANAWGAA、 AD’2 ：NGTCGASWGANAWGAACCCGGG；AD3：NGTASASWGTNAWCAA、 AD’3：NGTASASWGTNAWCAACCCGGG；AD4：STTGNTASTNCTNTGC、AD’4：STTGNTASTNCTNTGCCCCGGG ；AD5：AGWGNAGWANCAWAGG、 AD’5：AGWGNAGWANCAWAGGCCCGGG；AD6 ：TGWGNAGWANCASAGA、 AD’6：TGWGNAGWANCASAGACCCGGG.
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| CN106967738A (en) * | 2017-02-21 | 2017-07-21 | 三明学院 | A kind of Phenylalanine Ammonia-Lyase Gene from roxburgh anoectochilus terminal bud |
| CN109593783B (en) * | 2017-09-30 | 2024-05-24 | 中国科学院动物研究所 | Method for in vitro generation of circular nucleic acid molecules |
| CN111518941B (en) * | 2020-05-13 | 2021-05-11 | 四川农业大学 | Nucleic acid sequence for detecting corn plant AN1 and detection method thereof |
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