CN108588197A - A method of detection trace Single-stranded DNA fragments - Google Patents
A method of detection trace Single-stranded DNA fragments Download PDFInfo
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- CN108588197A CN108588197A CN201810613310.9A CN201810613310A CN108588197A CN 108588197 A CN108588197 A CN 108588197A CN 201810613310 A CN201810613310 A CN 201810613310A CN 108588197 A CN108588197 A CN 108588197A
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Abstract
A kind of method of detection trace Single-stranded DNA fragments provided by the invention, includes the following steps：Design Adapter pairs of cohesive end, including Adapter L and Adapter R；Wherein, Adapter L are used to capture the ends 5` of target DNA fragments, and Adapter R are used to capture the ends 3` of target DNA fragments；Cohesive end Adapter is added into the sample containing target DNA fragments to reaction, under the action of DNA Ligase, makes Adapter pairs to be connect with the cohesive end of target DNA fragments；After product purification, by PCR amplification up to the target dna after enrichment.Cohesive end Adapter of the present invention makes it obtain a large amount of enrichments the target dna for capturing low initial amount；For the Adapter to being used for DNA fragmentation enrichment, operating process is simple, can be quickly obtained a large amount of DNA fragmentation, and the sample preparation to solve the problems, such as the specific molecular Biological Detection field such as the NGS library constructions, chip hybridization, fluorescence immunoassay in forward position the most is difficult.
Description
Technical field
The present invention relates to molecular biosciences experimental technique fields, and in particular to a method of detection trace Single-stranded DNA fragments.
Background technology
With the development of bioscience technology, molecular biology experiment technology is also flourishing flourishing.The technology is in scientific experiment
Room, agricultural breeding, clinic diagnosis etc. play an important role.
In terms of scientific research, monoclonal technology of preparing so that researcher can easily obtain purpose product.In breeding side
The gene of one rush high yield is transferred to another species by clone, it is made to obtain high yield characteristics by face.Other laboratories then make
Characteristics such as a kind of agricultural crops are resisted cold, is drought-enduring, saline-alkali tolerant, etc., it is too numerous to enumerate.
During clinic diagnosis, emerging Protocols in Molecular Biology is also continued on.From initial PCR amplification, exempt from
Epidemic disease group technology, to fluorescent quantitative PCR technique, fluorecyte observational technique, finally arrive high throughput sequencing technologies and subsequently more
Add cutting edge technology etc..The update iteration of technology so that the amount of DNA of original input is fewer and fewer, and initial ug ranks are finally several
Hundred ng ranks.What is more, digital pcr can detect the exception of several copy numbers, and input amount is no more than 40ng.The input of DNA
The reduction of amount, the sample scope that can be used for detecting is more and more wider, and the DNA for sample segment extraction often occur excessively is measured less, can not
The case where meeting any type detection means.For such situation, present invention introduces a kind of cohesive end Adapter to design
And its application method, the target DNA fragments that can be measured with being enriched with simple, fast and easyly.
Invention content
Technical problem：In order to solve the defects of prior art, the present invention provides a kind of detection trace Single-stranded DNA fragments
Method.
Technical solution：A kind of method of detection trace Single-stranded DNA fragments provided by the invention, includes the following steps：
(1) Adapter pairs of cohesive end, including Adapter L and Adapter R are designed；Wherein, Adapter L are used for
The ends 5` of target Single-stranded DNA fragments are captured, Adapter R are used to capture the ends 3` of target Single-stranded DNA fragments；
(2) cohesive end Adapter is added into the sample of the Single-stranded DNA fragments containing target to reaction, in DNA Ligase
Under the action of, Adapter pairs connect with the cohesive end of target Single-stranded DNA fragments；
(3) after step (2) product purification, by PCR amplification up to the target Single-stranded DNA fragments after enrichment.
Preferably, the method for the detection trace Single-stranded DNA fragments, includes the following steps：
(1) detection of nucleic acids instrument is used to carry out Quality Control to Single-stranded DNA fragments；
(2) Single-stranded DNA fragments after 1ul Quality Controls are added into PCR pipe, add reaction reagent, the 20-37 in PCR instrument
30min is reacted at DEG C, reaction reagent is：
| Reaction reagent | Volume |
| Sample containing Single-stranded DNA fragments | 20ul |
| T4 Polymerase Buffer(10x) | 2.5ul |
| T4 PNK | 2ul |
| T4 DNA Ligase Buffer(10x) | 2.5ul |
| T7 DNA Ligase | 1ul |
| Adapter L | 2.5ul |
| Adapter R | 2.5ul |
| Water | Add to 50ul |
(3) product of step (2) the AMPure XP beads processes of 1.8 times of volumes are purified, water of the back dissolving in 23ul
In；PCR reaction systems are added, the circular response in PCR instrument；
Wherein, PCR reaction systems are：
| PCR reaction system components | Volume |
| Sample after back dissolving | 23ul |
| PCR Master Mix(2x) | 25ul |
| M13 F primer(10um) | 1ul |
| M13 R primer(10um) | 1ul |
| H2O | - |
Circular response program is：95 DEG C, 45s；Into cycle：98 DEG C, 20s；55 DEG C, 30s；72 DEG C, 30s, totally 25 are followed
Ring；72 DEG C, 1min；4 DEG C, ∞；
(4) product of step (3) the AMPure XP beads processes of 1.8 times of volumes are purified, you can；
SEQ ID NO.5:AATGA/ideoxyU/ACGGCGACCACCGAGATC/ideoxyU/ACACTCTTTCCC/
ideoxyU/ ACACGACNNNNNN；
SEQ ID NO.6:GTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT；
SEQ ID NO.7:NNNNNNcaagcagaagacggca/ideoxyU/acgagatTGCATGACgtgactggagt
/ ideoxyU/cagacgtgt；
SEQ ID NO.8:GTTCGTCTTCTGCCGTATGCTCTAACGTACTGCACTGACCTCAAGTCTGCAC。
Advantageous effect：Cohesive end Adapter of the present invention makes it obtain big the target dna for capturing low initial amount
Amount enrichment；For the Adapter to being used for DNA fragmentation enrichment, operating process is simple, can be quickly obtained a large amount of DNA fragmentation, to
Solve the sample preparation in the specific molecular Biological Detection field of the NGS library constructions, chip hybridization, fluorescence immunoassay in forward position etc. the most
Difficult problem.
Specifically, the present invention compared with the existing technology, have the advantages that it is following prominent：
(1) present invention is directed to DNA, no particular/special requirement, and applicability is wide；
(2) method is simple and feasible, is easy to practical execution；
(3) more enough DNA samples for downstream research can be obtained.
Description of the drawings
Fig. 1 is the design display diagram of left side Adapter of the present invention, and Adapter has short complementary pairing in figure
Region, and the ends 5` of one DNA chain protrude cohesive end, for capturing target DNA fragments；
Fig. 2 is the design display diagram of right side Adapter of the present invention, and Adapter has short complementary pairing in figure
Region, and the ends 3` of one DNA chain protrude cohesive end, for capturing target DNA fragments, with left side Adapter
Cooperation uses in pairs；
Fig. 3 is right side of the present invention and right side Adapter to capturing the principle display diagrams of target DNA fragments, in figure
Right side and right side Adapter can be bonded directly to the both ends of the DNA fragmentation of single stranded, under the action of ligase, make right side
The both ends of target DNA fragments are connected to the segment on the Adapter of right side, product can be obtained a large amount of targets by PCR amplification
DNA fragmentation；
Fig. 4 is testing result figure of the target DNA fragments before enrichment in the specific embodiment of the invention 1, and such as middle displaying should
Target DNA fragments size is in 214bp, and concentration is in 6.84ng/ul；
Fig. 5 is the testing result figure of target DNA fragments after the enrichment in the specific embodiment of the invention 1, and such as middle displaying should
Target DNA fragments size is in 285bp, and concentration is in 8.64ng/ul；
Fig. 6 is testing result figure of the target DNA fragments before enrichment in the specific embodiment of the invention 2, and such as middle displaying should
Target DNA fragments size is in 169bp, and concentration is in 0.65ng/ul；
Fig. 7 is the testing result figure of target DNA fragments after the enrichment in the specific embodiment of the invention 2, and such as middle displaying should
Target DNA fragments size is in 289bp, and concentration is in 21.64ng/ul.
Specific implementation mode
The content that following embodiment further illustrates the present invention, but should not be construed as limiting the invention.Without departing substantially from
In the case of spirit of that invention and essence, to modifications or substitutions made by the method for the present invention, step or condition, the present invention is belonged to
Range.
Unless otherwise specified, the conventional means that technological means used in embodiment is well known to those skilled in the art；
Reagent used in embodiment is commercial goods.Detection of nucleic acids instrument and reagent are the 2100 nucleic acid inspection that Agilent companies produce
Survey instrument and related reagent.Agents useful for same is the product that NEB companies produce, such as following table：
Purified reagent is the AMPure XP beads of Beckman Culter companies.
The enrichment of 1 short dna segment of embodiment
Trace Single-stranded DNA fragments are detected, are included the following steps：
One, Adapter pairs of cohesive end, including Adapter L and Adapter R are designed；Wherein, Adapter L are used for
The ends 5` of target Single-stranded DNA fragments are captured, Adapter R are used to capture the ends 3` of target Single-stranded DNA fragments；
Adapter sequence designs based on M13F and M13R primer sequences：
1, in long sequence, Ad-R and Ad-L two sequences are added the U bases of modification, and block are added at the ends 5`
It is closed；
2, the preparation method of M13R-Adapter-R：By Ad-R and M13R mixed in equal amounts, slowly anneal after high temperature, 0.1
℃/s。
3, the preparation method of M13F-Adapter-L：By Ad-L and M13F mixed in equal amounts, slowly anneal after high temperature, 0.1
℃/s。
Two, cohesive end Adapter is added into the sample of the Single-stranded DNA fragments containing target to reaction, in DNA Ligase
Under the action of, Adapter pairs connect with the cohesive end of target Single-stranded DNA fragments；
1. short dna segment is carried out Quality Control using 2100 detection of nucleic acids instrument, as a result such as Fig. 4；
2. the short DNA fragmentations of 1ul are added in PCR pipe；
3. following reagent is added into PCR pipe：
4. in PCR instrument, 27 DEG C of reaction 30min；
Three, after step 2 product purification, by PCR amplification up to the target Single-stranded DNA fragments after enrichment.
1. the above-mentioned product AMPure XP beads processes of 1.8 times of volumes are purified, H of the back dissolving in 23ul2In O；
2. the following PCR reaction systems of addition：
| Adding ingredient | Volume |
| Step (5) product | 23ul |
| PCR Master Mix(2x) | 25ul |
| M13 F primer(10um) | 1ul |
| M13 R primer(10um) | 1ul |
| H2O | - |
3. in PCR instrument, 95 DEG C, 45s；(98 DEG C, 20s；55 DEG C, 30s；72 DEG C, 30s；) 72 DEG C, 1min；4 DEG C, ∞；
Bracket internal program runs 25 cycles；
4. the above-mentioned PCR product AMPure XP beads processes of 1.8 times of volumes are purified, H of the back dissolving in 25ul2In O；
5. carrying out concentration mensuration, result 6.84ng/ul to final product using Qubit, and 1 μ l are taken, with 2100 nucleic acid
Detector carries out segment ranges detection, preserves picture, as a result sees Fig. 5.
As shown in figure 4, before using the technology, DNA fragmentation mainly using 214bp as the segment ranges of main peak, at both ends plus
Adapter based on upper M13F and M13R sequences is to later, target DNA fragment length increased.Target DNA fragment increases
Part be DNA fragmentation both ends M13F and M13R sequences, 18bp+18bp=36bp ultimately becomes 214bp+36np=
250bp, it is contemplated that for 2100bp to the systematic error in DNA quality control process, it is that 255bp is which, which obtains target fragment result,
The product of main peak (shown in Fig. 5).
The enrichment of 2 cfDNA segments of embodiment
Trace Single-stranded DNA fragments are detected, are included the following steps：
One, Adapter pairs of cohesive end, including Adapter L and Adapter R are designed；Wherein, Adapter L are used for
The ends 5` of target Single-stranded DNA fragments are captured, Adapter R are used to capture the ends 3` of target Single-stranded DNA fragments；
Adapter sequence designs based on P5 and P7 primer sequences：
1, in long sequence, Ad-P5 and Ad-P7 two sequences are added the U bases of modification, and are added at the ends 5`
Block is closed；
2, the preparation of P5-Adapter-R：By Ad-P5 and P5 mixed in equal amounts, slowly anneal after high temperature, 0.1 DEG C/s；
3, the preparation of P7-Adapter-L：By Ad-P7 and P7 mixed in equal amounts, slowly anneal after high temperature, 0.1 DEG C/s；It is another
End connector is prepared with same method.
Two, cohesive end Adapter is added into the sample of the Single-stranded DNA fragments containing target to reaction, in DNA Ligase
Under the action of, Adapter pairs connect with the cohesive end of target Single-stranded DNA fragments；
1. short dna segment is carried out Quality Control using 2100 detection of nucleic acids instrument, as a result such as Fig. 6；
2. the short DNA fragmentations of 1ul are added in PCR pipe；
3. following reagent is added into PCR pipe：
| Adding ingredient | Volume |
| Sample containing DNA fragmentation | 20ul |
| T4 Polymerase Buffer(10x) | 2.5ul |
| T4 PNK | 2ul |
| T4 DNA Ligase Buffer(10x) | 2.5ul |
| T7 DNA Ligase | 1ul |
| P5-Adapter-L(28uM) | 2.5ul |
| P7-Adapter-R(28uM) | 2.5ul |
| H2O | Up to 50ul |
4. in PCR instrument, 27 DEG C of reaction 30min；
Three, after step 2 product purification, by PCR amplification up to the target Single-stranded DNA fragments after enrichment.
1. the above-mentioned product AMPure XP beads processes of 1.8 times of volumes are purified, H of the back dissolving in 23ul2In O；
2. the following PCR reaction systems of addition：
| Adding ingredient | Volume |
| Step (5) product | 23ul |
| PCR Master Mix(2x) | 25ul |
| P5 primer(10um) | 1ul |
| P7 primer(10um) | 1ul |
| H2O | - |
3. in PCR instrument, 95 DEG C, 45s；(98 DEG C, 20s；55 DEG C, 30s；72 DEG C, 30s；) 72 DEG C, 1min；4 DEG C, ∞；
Bracket internal program runs 25 cycles；
4. the above-mentioned PCR product AMPure XP beads processes of 1.8 times of volumes are purified, H of the back dissolving in 25ul2In O；
5. carrying out concentration mensuration, result 21.07ng/ul to final product using Qubit, and 1 μ l are taken, with 2100 nucleic acid
Detector carries out segment ranges detection, as a result sees Fig. 7.
As shown in fig. 6, before using the technology, DNA fragmentation mainly using 169bp as the segment ranges of main peak, at both ends plus
Adapter based on upper P5 and P7 sequences is to later, target DNA fragment length increased.The increased portion of target DNA fragment
It is divided into P5 the and P7 sequences at DNA fragmentation both ends, 62bp+62bp=124bp ultimately becomes 169bp+124np=293bp, considers
To 2100bp to the systematic error in DNA quality control process, it is the product that 289bp is main peak which, which obtains target fragment result,
(shown in Fig. 7).
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (4)
1. a kind of method of detection trace Single-stranded DNA fragments, it is characterised in that：Include the following steps：
(1) Adapter pairs of cohesive end, including Adapter L and Adapter R are designed；Wherein, Adapter L are for capturing
The ends 5` of target Single-stranded DNA fragments, Adapter R are used to capture the ends 3` of target Single-stranded DNA fragments；
(2) cohesive end Adapter is added into the sample of the Single-stranded DNA fragments containing target to reaction, in the work of DNA Ligase
Under, Adapter pairs connect with the cohesive end of target Single-stranded DNA fragments；
(3) after step (2) product purification, by PCR amplification up to the target Single-stranded DNA fragments after enrichment.
2. a kind of method of detection trace Single-stranded DNA fragments according to claim 1, it is characterised in that：Including following step
Suddenly：
(1) detection of nucleic acids instrument is used to carry out Quality Control to Single-stranded DNA fragments；
(2) Single-stranded DNA fragments after 1ul Quality Controls are added into PCR pipe, reaction reagent are added, in PCR instrument at 20-37 DEG C
30min is reacted, reaction reagent is：
(3) product of step (2) the AMPure XP beads processes of 1.8 times of volumes are purified, and back dissolving is in the water of 23ul；Again
PCR reaction systems, the circular response in PCR instrument is added；
Wherein, PCR reaction systems are：
Circular response program is：95 DEG C, 45s；Into cycle：98 DEG C, 20s；55 DEG C, 30s；72 DEG C, 30s, totally 25 recycle；72
DEG C, 1min；4 DEG C, ∞；
(4) product of step (3) the AMPure XP beads processes of 1.8 times of volumes are purified, you can.
3. a kind of method of detection trace Single-stranded DNA fragments according to claim 1 or 2, it is characterised in that：It is described
Adapter R are M13R-Adapter-R, and preparation method is：By Ad-R and M13R mixed in equal amounts, slowly anneal after high temperature,
0.1 DEG C/s, wherein the amino acid sequence of Ad-R is as shown in SEQ ID NO.1, the amino acid sequence such as SEQ ID NO.2 of M13R
It is shown；The Adapter L are M13F-Adapter-L, and preparation method is：By Ad-L and M13F mixed in equal amounts, delay after high temperature
Slow annealing, 0.1 DEG C/s, wherein the amino acid sequence of Ad-L is as shown in SEQ ID NO.3, the amino acid sequence such as SEQ of M13F
Shown in ID NO.4；
SEQ ID NO.1:CAGGAAACAGCTA/ideoxyU/GACCNNNNNNNN；
SEQ ID NO.2:GGTCATAGCTGTTTCCTG；
SEQ ID NO.3:NNNNNNNNAC/ideoxyU/GGCCGTCG/ideoxyU/TTTACA；
SEQ ID NO.4:TGTAAAACGACGGCCAGT。
4. a kind of method of detection trace Single-stranded DNA fragments according to claim 1 or 2, it is characterised in that：It is described
Adapter R are P5-Adapter-R, and preparation method is：By Ad-P5 and P5 mixed in equal amounts, slowly anneal after high temperature, 0.1
DEG C/s, wherein the amino acid sequence of Ad-P5 is as shown in SEQ ID NO.5, and the amino acid sequence of P5 is as shown in SEQ ID NO.6；
The Adapter L are P7-Adapter-L, and preparation method is：By Ad-P7 and P7 mixed in equal amounts, slowly anneal after high temperature,
0.1 DEG C/s, wherein the amino acid sequence of Ad-P7 is as shown in SEQ ID NO.7, the amino acid sequence such as SEQ ID NO.8 institutes of P7
Show；
SEQ ID NO.5:AATGA/ideoxyU/ACGGCGACCACCGAGATC/ideoxyU/ACACTCTTTCCC/
ideoxyU/ACACGACNNNNNN；
SEQ ID NO.6:GTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT；
SEQ ID NO.7:NNNNNNcaagcagaagacggca/ideoxyU/acgagatTGCATGACgtgactggagt/
ideoxyU/cagacgtgt；
SEQ ID NO.8:GTTCGTCTTCTGCCGTATGCTCTAACGTACTGCACTGACCTCAAGTCTGCACA。
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Cited By (1)
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| CN110514631A (en) * | 2019-08-12 | 2019-11-29 | 南京医科大学 | A kind of highly sensitive, rapid quantitative detection T4 PNK enzyme method |
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