CN106011230A - Primer composition for detecting fragmentized DNA target area and application thereof - Google Patents
Primer composition for detecting fragmentized DNA target area and application thereof Download PDFInfo
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Abstract
The invention discloses a primer composition for detecting a fragmentized DNA target area and application thereof. The primer composition comprises a first primer group and a second primer group; the first primer group contains a first universal primer and a second universal primer, and a target point of the first universal primer and a target point of the second universal primer are located at the two ends of the target area and used for forming a 5' end and a 3' end of a sequencing library respectively; the second primer group comprises a first specific primer group and a second specific primer group. By conducting amplification enrichment on the fragmentized DNA target area through the primer composition, the adaptive range of primer amplification can be significantly widened, the effective template quantity of primer amplification can be significantly increased, then sequencing detection is conducted on an enrichment product, and the fragmentized DNA detection sensitivity can be significantly improved.
Description
Technical field
The present invention relates to fragmentation DNA detection technical field, in particular it relates to be used for detecting fragmentation DNA target area
Primer composition and application.
Background technology
When scientific research personnel carries out biological study, need to extract nucleic acid (DNA, or RNA) from various types of samples.Its
In, body fluid sample contains the DNA of fragmentation, and it is important detecting step that the DNA in this sample carries out Biological Detection.Such as
Plasma dna, is extracellular DNA in blood, presented in nucleosome (DNA-protein complex).This kind of fragmentation
DNA length is tens to hundreds of base pair (main peak is 166bp).This kind of fragmentation DNA is generally discharged extremely by a small amount of apoptotic cell
In blood circulation, content is extremely low, and existing PCR method is difficult to authentic communication be detected.
It addition, fossil all has the value of uniqueness in multi-disciplinary research is permitted in biological evolution, heredity, form, classification etc..
Owing to the evolution of DNA level occupies very important status in theory of biological evolution, and fossil DNA is to be directly acquainted with DNA to evolve
What of process was important is also likely to be unique approach, and therefore the research in this field is by important for become molecular evolution opinion one
Aspect.But fossil DNA is in long forming process, DNA has degraded to the least fragment, and content is the dilutest simultaneously
Few, existing detection method does not often reach the testing requirement of scientific research personnel.
Paraffin embedding (FFPE) sample solves the problem that fresh sample preserves for a long time, but sample is solid through formalin
After fixed, paraffin embedding, DNA is readily formed crosslinking fragmentation, and usually tens to thousand of base pairs.Scientific research personnel is difficult to
High-quality DNA is obtained for highly sensitive detection from this kind of material.
Criminal investigation sample be Public Security Organs for the important evidence solved a case, during solving a case, play important role.But
It is that criminal investigation sample generally has degraded in various degree because of the difference of local environment, criminal investigation sample DNA.Criminal investigation sample DNA simultaneously
It is frequently not the DNA of pure single individuality, but is mixed with the mixture of other individual DNA, and real significant DNA's is mixed
Composition and division in a proportion example the most extremely low (0.1%), but, existing detection method is extremely difficult to the highest detection sensitivity.
Thus, current fragmentation DNA detection method still haves much room for improvement.
Summary of the invention
It is contemplated that at least solve one of technical problem present in prior art.To this end, one object of the present invention
It is to propose a kind of DNA utilization rate fragmentation DNA detection method being particularly suited for trace sample high, highly sensitive.
It should be noted that the present invention is following discovery based on inventor and work and completes:
For fragmentation DNA, traditional detection method is that detection region is designed a pair aspectant primer (forward primer
And reverse primer), by PCR (polymerase chain reaction), this region is carried out index amplification, finally by instrument to this amplification
Product detects.Can not occur the situation of breakaway poing in the guiding region of traditional PCR detection technique, otherwise, PCR experiment will
Failure.Due to fragmentation DNA, the efficiency that traditional PCR technique certainly will be caused to may utilize template reduces.
Specifically, inventor studies discovery, and the method for traditional detection fragmentation DNA is mainly passed through to treat detection region and carried out
PCR amplification detects, because PCR primer is to design in the both sides in region to be detected, therefore it is required that region to be detected has kept
Whole, but fragmentation DNA is random fracture to be produced, and most of fragmentation DNA are incomplete, accordingly, it is capable to as pcr template
The fragmentation DNA quantity used is few, and PCR is difficult to detect.And then, inventor creatively uses specific primer group in the same direction
Fragmentation DNA is enriched with, thus adds the subject range of primer amplification, effective template amount, and make fragmentation DNA's
Detection sensitivity is greatly improved, and efficiently solves a current technology difficult problem.
And then, in a first aspect of the present invention, the present invention proposes a kind of for detecting drawing of fragmentation DNA target area
Compositions.According to embodiments of the invention, this Primer composition includes:
First primer sets, described first primer sets comprises the first universal primer and the second universal primer, described first general
The target spot of primer and described second universal primer is positioned at the two ends of described target area, is respectively used to constitute 5 ' ends of sequencing library
With 3 ' ends；And
Second primer sets, described second primer sets includes the first specific primer group and the second specific primer group,
Wherein,
Described first specific primer group and described second specific primer group all comprise n bar target regiospecificity and draw
Thing, and described first specific primer group and described second specific primer group must be fulfilled for following condition:
(1) each specific primer of described first specific primer group and described second specific primer group prolongs the most in the same direction
Stretching, target spot is respectively positioned on 5 ' ends or the 3 ' ends of described fragmentation DNA target area；
(2) arbitrary integer of n=1-5, as a example by n=5, if each specific primer of described first specific primer group divides
It is not: N1、N2、N3、N4、N5, each specific primer of described second specific primer group is respectively as follows: M1、M2、M3、M4、M5, with spy
Distance between target spot and the target area of specific primer is criterion, described first specific primer group and described second spy
Distance relation between each specific primer of specific primer group is:
N1>N2>N3>N4>N5；
M1>M2>M3>M4>M5；
N1>M1；
N2>M2；
N3>M3；
N4>M4；And
N5>M5,
Wherein, the target spot of each specific primer of described first specific primer group and described second specific primer group,
With the two ends that the target spot of described first universal primer lays respectively at described target area, with the target spot position of described second universal primer
Same end in described target area.
It is surprisingly found by the inventors that, the Primer composition of the present invention can be effective to detect fragmentation DNA target area.
Specifically, the Primer composition utilizing the present invention carries out amplification enrichment to the target area of fragmentation DNA, it is possible to significantly improves and draws
The subject range of thing amplification and effective template amount, and then enriched product is carried out order-checking detection, it is possible to significantly improve fragmentation DNA
Detection sensitivity.Thus, the Primer composition of the present invention is particularly suited for the fragmentation DNA detection of trace sample.
In a second aspect of the present invention, the present invention proposes a kind of detection library, target area building fragmentation DNA
Method.According to embodiments of the invention, the method utilizes foregoing Primer composition, described broken by PCR amplification enrichment
The sequence of sheet DNA target area.
According to embodiments of the invention, the method is utilized can effectively to build the detection library, target area of fragmentation DNA.
Further, the method utilizes aforesaid Primer composition that the target area of fragmentation DNA carries out amplification enrichment such that it is able to notable
Improve the subject range of primer amplification and effective template amount, it is thus achieved that enriched product i.e. sequencing library after high-flux sequence,
Accurately and reliably, the detection sensitivity of fragmentation DNA is high, and favorable repeatability for target area sequencing result.Further, the method is especially
The target area sequencing library of the fragmentation DNA being suitable to trace sample builds.
In a third aspect of the present invention, the present invention proposes a kind of side determining fragmentation DNA target area sequence information
Method.According to embodiments of the invention, the method includes:
The method in the detection library, target area according to foregoing structure fragmentation DNA, builds described fragmentation DNA
Detection library, target area；
Is checked order in the detection library, target area of described fragmentation DNA, in order to obtain sequencing result；And
Based on described sequencing result, determine the sequence information of described fragmentation DNA target area.
Inventor finds, utilizes the method can effectively determine fragmentation DNA target area sequence information.Further, based on
The Primer composition of the aforementioned present invention of utilization carries out amplification enrichment to the target area of fragmentation DNA, thus notable raising is drawn
The subject range of thing amplification and effective template amount, and it is effectively increased expanding effect specific amplification, and then the enrichment obtained is produced
Thing is after high-flux sequence, and accurately and reliably, the detection sensitivity of fragmentation DNA is high, and repeatable for target area sequencing result
Property is good.Further, the method is particularly suited for the fragmentation DNA detection of trace sample, all anticipates in terms of scientific research and production application
Justice is great, is suitable to promote.
In a fourth aspect of the present invention, the present invention proposes a kind of dress building detection library, fragmentation DNA target area
Put.According to embodiments of the invention, this device is provided with foregoing Primer composition, and described device includes:
Oneth PCR amplification unit, a described PCR amplification unit is used for utilizing the first specific primer group and first general
Primer carries out a PCR amplification to described fragmentation DNA, in order to obtain the first pcr amplification product；
2nd PCR amplification unit, described 2nd PCR amplification unit is connected with a described PCR amplification unit, is used for utilizing
Second specific primer group and the first universal primer carry out the 2nd PCR amplification to described first pcr amplification product, in order to obtain the
Two pcr amplification products；And
3rd PCR amplification unit, described 3rd PCR amplification unit is connected with described 2nd PCR amplification unit, is used for utilizing
First universal primer and the second universal primer carry out the 3rd PCR amplification to described second pcr amplification product, in order to obtain the 3rd
Pcr amplification product, described 3rd pcr amplification product constitutes the detection library, target area of described fragmentation DNA.
According to embodiments of the invention, this device utilizes aforesaid Primer composition to enter the target area of fragmentation DNA
Row amplification enrichment such that it is able to significantly improve the subject range of primer amplification and effective template amount, it is thus achieved that enriched product i.e. survey
Preface storehouse is good for the specificity of target area, and then after high-flux sequence, target area sequencing result is accurately and reliably, broken
The detection sensitivity of sheet DNA is high, and favorable repeatability.Further, this device is particularly suited for the mesh of fragmentation DNA of trace sample
Mark region sequencing library builds.
In a fifth aspect of the present invention, the present invention propose a kind of determine fragmentation DNA target area sequence information be
System.According to embodiments of the invention, this system includes:
The device in detection library, foregoing structure fragmentation DNA target area, for building described fragmentation DNA's
Detection library, target area；
Sequencing device, described sequencing device is connected with the device in detection library, described structure fragmentation DNA target area, uses
In being checked order in the detection library, target area of described fragmentation DNA, in order to obtain sequencing result；And
Sequence Determination Means, described Sequence Determination Means is connected with described sequencing device, is used for based on described sequencing result,
Determine the sequence information of described fragmentation DNA target area.
It is surprisingly found by the inventors that, this system utilizes the Primer composition of the present invention to enter the target area of fragmentation DNA
Row amplification enrichment, thus the notable subject range improving primer amplification and effective template amount, and it is effectively increased expanding effect
Specific amplification, and then the enriched product obtained is after high-flux sequence, can not only effectively determine fragmentation DNA target area
The sequence information in territory, and sequencing result is accurately and reliably, detection sensitivity is high, favorable repeatability.Further, this system is particularly suited for micro-
The fragmentation DNA detection of amount sample, the most significant in terms of scientific research and production application, be suitable to promote.
The additional aspect of the present invention and advantage will part be given in the following description, and part will become from the following description
Obtain substantially, or recognized by the practice of the present invention.
Accompanying drawing explanation
Above-mentioned and/or the additional aspect of the present invention and advantage are from combining the accompanying drawings below description to embodiment and will become
Substantially with easy to understand, wherein:
Fig. 1 shows the design principle schematic diagram of the specific primer extended in the same direction of the present invention；
Fig. 2 shows that the Primer composition of the present invention carries out the principle schematic of mutational site detection；
Fig. 3 shows that the Primer composition of the present invention carries out the principle schematic of gene fusion detection；
Fig. 4 shows the structure of the device in detection library, structure fragmentation DNA target area according to embodiments of the present invention
Schematic diagram；
Fig. 5 shows that the structure of the system determining fragmentation DNA target area sequence information according to embodiments of the present invention is shown
It is intended to；
Fig. 6 shows the electrophoresis detection result of the purified product of embodiment 1；And
Fig. 7 shows the electrophoresis detection result of the purified product of embodiment 2.
Detailed description of the invention
Embodiments of the invention are described below in detail.The embodiments described below is exemplary, is only used for explaining this
Bright, and be not considered as limiting the invention.
Primer composition
In a first aspect of the present invention, the present invention proposes a kind of primer sets for detecting fragmentation DNA target area
Compound.According to embodiments of the invention, this Primer composition includes:
First primer sets, described first primer sets comprises the first universal primer and the second universal primer, described first general
The target spot of primer and described second universal primer is positioned at the two ends of described target area, is respectively used to constitute 5 ' ends of sequencing library
With 3 ' ends；And
Second primer sets, described second primer sets includes the first specific primer group and the second specific primer group,
Wherein,
Described first specific primer group and described second specific primer group all comprise n bar target regiospecificity and draw
Thing, and described first specific primer group and described second specific primer group must be fulfilled for following condition:
(1) each specific primer of described first specific primer group and described second specific primer group prolongs the most in the same direction
Stretching, target spot is respectively positioned on 5 ' ends or the 3 ' ends of described fragmentation DNA target area；
(2) arbitrary integer of n=1-5, as a example by n=5, if each specific primer of described first specific primer group divides
It is not: N1、N2、N3、N4、N5, each specific primer of described second specific primer group is respectively as follows: M1、M2、M3、M4、M5, with spy
Distance between target spot and the target area of specific primer is criterion, described first specific primer group and described second spy
Distance relation between each specific primer of specific primer group is:
N1>N2>N3>N4>N5；
M1>M2>M3>M4>M5；
N1>M1；
N2>M2；
N3>M3；
N4>M4；And
N5>M5,
Wherein, the target spot of each specific primer of described first specific primer group and described second specific primer group,
With the two ends that the target spot of described first universal primer lays respectively at described target area, with the target spot position of described second universal primer
Same end in described target area.
It is surprisingly found by the inventors that, the Primer composition of the present invention can be effective to detect fragmentation DNA target area.
Specifically, the Primer composition utilizing the present invention carries out amplification enrichment to the target area of fragmentation DNA, it is possible to significantly improves and draws
The subject range of thing amplification and effective template amount, and then enriched product is carried out order-checking detection, it is possible to significantly improve fragmentation DNA
Detection sensitivity.Thus, the Primer composition of the present invention is particularly suited for the fragmentation DNA detection of trace sample.
Wherein, the Primer composition utilizing the present invention carries out expanding enrichment and includes the target area of fragmentation DNA: utilize
First specific primer group and the first universal primer carry out a PCR amplification to described fragmentation DNA, in order to obtain a PCR
Amplified production；Utilize the second specific primer group and the first universal primer that described first pcr amplification product carries out the 2nd PCR to expand
Increase, in order to obtain the second pcr amplification product；And utilize the first universal primer and the second universal primer that described 2nd PCR is expanded
Volume increase thing carries out the 3rd PCR amplification, in order to obtain the 3rd pcr amplification product, then the 3rd pcr amplification product is enrichment product
Thing, the detection library, target area of follow-up composition described fragmentation DNA.
For convenience of understanding, now the principle that the present invention relates to is explained:
For the specific primer of the present invention, in brief, the specific primer group of the present invention is two and extends in the same direction
Primer sets.Wherein, Fig. 1 shows the design principle of the present invention: assume that genome uniformly ruptures, and ※ is point mutation to be checked, inserts
Enter, deletion segment；A is conventional primer, and available template is the fragmentation DNA of heavy line；B is the primer of the present invention, can profit
Template be the fragmentation DNA that heavy line adds fine line；As can be seen here, the primer available template number of present invention design is remote
Higher than traditional method.
Fig. 2 shows mutational site Cleaning Principle.Wherein, b1 is the specific primer extended in the same direction, and ※ is point to be checked
Sudden change, insertion, deletion segment, solid line boxes is universal joint, and b2 is universal primer.It follows that utilize, the present invention's is special
Property primer sets carries out PCR amplification, can effectively utilize all containing extending the fragmentation DNA of primer in the same direction as template, be higher than
Normal PCR.
Fig. 3 shows the Cleaning Principle for gene fusion.Wherein, left side is A gene, and right side is 1 B gene, A gene and B
Gene junction is position of fusion, and b1 is the specific primer extended in the same direction, and solid line boxes is universal joint, and b2 is general drawing
Thing.It follows that utilize the specific primer group of the present invention to carry out PCR amplification, can effectively utilize all containing extending in the same direction
The fragmentation DNA of primer is as template, higher than normal PCR.
That is, utilize the specific primer group of the present invention to carry out PCR amplification, because available DNA can be significantly improved
Template amount such that it is able to improve the sensitivity of detection.
According to embodiments of the invention, for each specific primer group, its each specific primer comprised is based on base
Because the order on group position is spaced 0-50 successively to base pair.Thus, amplification efficiency is high.
According to embodiments of the invention, N1With M1Corresponding genomic locations has overlap, N2With M2Corresponding genomic locations
There are overlap, N3 and M3Corresponding genomic locations has overlap, N4With M4Corresponding genomic locations has overlap, N5With M5Corresponding base
Because there is overlap group position, preferably overlapping 0-20 base pair.Thus, expanding effect is good.
According to embodiments of the invention, the G/C content of each specific primer is 30%-70%, Tm value and is 55-68 DEG C.By
This, expanding effect is good.
It should be noted that the particular sequence of universal primer is not particularly limited, can order-checking platform based on follow-up use
Select, namely use with order-checking platform with the use of two sequencing primers can (respectively constitute sequencing library 5 ' end and 3 '
End).According to some specific embodiments of the present invention, two universal primers are respectively as follows:
First universal primer: CAAGCAGAAGACGGCATACGA (SEQ ID NO:1)；
Second universal primer: AATGATACGGCGACCACCGA (SEQ ID NO:2).
According to embodiments of the invention, described fragmentation DNA target area is EGFR gene 19 exon, and n=2,
Each specific primer of described first specific primer group is respectively as follows:
N1: TGCCAGTTAACGTCTTCCTTC (SEQ ID NO:3),
N2: ATAGGGACTCTGGATCCCAG (SEQ ID NO:4)；And
Each specific primer of described second specific primer group is respectively as follows:
M1: GTCTTCCTTCTCTCTCTGTCATAG (SEQ ID NO:5)
M2: TGGATCCCAGAAGGTGAGAAAGTTA (SEQ ID NO:6).Thus, primer based on present invention combination
Thing, it is possible to the effectively EGFR gene 19 exon region to fragmentation DNA carries out amplification enrichment and order-checking detection, and available
Template amount is big, amplification efficiency is high, specificity carefully.
According to embodiments of the invention, described fragmentation DNA target area is EGFR gene 20 exon, and n=2,
Each specific primer of described first specific primer group is respectively as follows:
N1: CCAGGAAGCCTACGTGATGGC (SEQ ID NO:7),
N2: TGGGCATCTGCCTCACCTCC (SEQ ID NO:8)；And
Each specific primer of described second specific primer group is respectively as follows:
M1: GATGGCCAGCGTGGACAACCCCCA (SEQ ID NO:9)
M2: CCTCACCTCCACCGTGCAGCTCAT (SEQ ID NO:10).Thus, primer based on present invention combination
Thing, it is possible to the effectively EGFR gene 20 exon region to fragmentation DNA carries out amplification enrichment and order-checking detection, and available
Template amount is big, amplification efficiency is high, specificity is good.
According to embodiments of the invention, the samples sources of fragmentation DNA is not particularly limited.Some tools according to the present invention
Body example, described fragmentation DNA is selected from plasma dna, urine DNA, perspiration DNA, saliva DNA, seminal fluid DNA, hydrothorax DNA, abdomen
At least one water DNA, faeces DNA, fossil DNA, paraffin embedding DNA and criminal investigation sample DNA.
According to embodiments of the invention, the target area of described fragmentation DNA comprise selected from point mutation, insert, lack, base
Because of merge sudden change at least one.Thus, after utilizing the sequence of Primer composition amplification enrichment target area of the present invention, enrichment
Product can be effective to detect fragmentation DNA target area point mutation, insert, lack, gene fusion sudden change.
The application of Primer composition
In a second aspect of the present invention, the present invention proposes a kind of detection library, target area building fragmentation DNA
Method.According to embodiments of the invention, the method utilizes foregoing Primer composition, described broken by PCR amplification enrichment
The sequence of sheet DNA target area.
According to embodiments of the invention, the method is utilized can effectively to build the detection library, target area of fragmentation DNA.
Further, the method utilizes aforesaid Primer composition that the target area of fragmentation DNA carries out amplification enrichment such that it is able to notable
Improve the subject range of primer amplification and effective template amount, it is thus achieved that enriched product i.e. sequencing library after high-flux sequence,
Accurately and reliably, the detection sensitivity of fragmentation DNA is high, and favorable repeatability for target area sequencing result.Further, the method is especially
The target area sequencing library of the fragmentation DNA being suitable to trace sample builds.
According to the concrete example of the present invention, said method comprising the steps of:
Utilize the first specific primer group and the first universal primer that described fragmentation DNA carries out a PCR amplification, in order to
Obtain the first pcr amplification product；
Utilize the second specific primer group and the first universal primer that described first pcr amplification product carries out the 2nd PCR to expand
Increase, in order to obtain the second pcr amplification product；And
Utilize the first universal primer and the second universal primer that described second pcr amplification product carries out the 3rd PCR amplification, with
Just obtaining the 3rd pcr amplification product, described 3rd pcr amplification product constitutes the detection library, target area of described fragmentation DNA.
Thereby, it is possible to effectively build the detection library, target area of fragmentation DNA, and Library Quality is good, be used for checking order detection time sensitive
Degree height, it is thus achieved that sequencing result accurately and reliably.
According to embodiments of the invention, before carrying out a described PCR amplification, farther include: to described fragmentation
DNA carries out end reparation successively, 3 ' ends add poly adenine tail and joint connects.
According to embodiments of the invention, farther include to be purified the product of each step.Thus, it is thus achieved that library
Quality is good, the carrying out of follow-up order-checking detection.
According to embodiments of the invention, utilize AmpliTaq360PCR Master Mix carries out a described PCR
Amplification and described 2nd PCR amplification.Thus, expanding effect is good.
According to embodiments of the invention, the response procedures of a described PCR amplification is: 95 DEG C 10 minutes；20 circulations: 95
DEG C 30 seconds, 62 DEG C 30 seconds, 72 DEG C 1 minute；72 DEG C 7 minutes；4 DEG C of preservations.Thus, expanding effect is good.
According to embodiments of the invention, the response procedures of described 2nd PCR amplification is: 95 DEG C 10 minutes；15 circulations: 95
DEG C 30 seconds, 62 DEG C 30 seconds, 72 DEG C 1 minute；72 DEG C 7 minutes；4 DEG C of preservations.Thus, expanding effect is good.
Wherein, the samples sources of fragmentation DNA is not particularly limited.According to some embodiments of the present invention, described fragment
Changing DNA is selected from plasma dna, urine DNA, perspiration DNA, saliva DNA, seminal fluid DNA, hydrothorax DNA, ascites DNA, faeces DNA, change
At least one stone DNA, paraffin embedding DNA and criminal investigation sample DNA.
According to embodiments of the invention, the target area of described fragmentation DNA comprise selected from point mutation, insert, lack, base
Because of merge sudden change at least one.Thus, the fragmentation DNA target area sequencing library obtained is built, it is possible to be effective to inspection
Survey target area point mutation, insert, lack, gene fusion sudden change.
In a third aspect of the present invention, the present invention proposes a kind of side determining fragmentation DNA target area sequence information
Method.According to embodiments of the invention, the method includes:
The method in the detection library, target area according to foregoing structure fragmentation DNA, builds described fragmentation DNA
Detection library, target area；
Is checked order in the detection library, target area of described fragmentation DNA, in order to obtain sequencing result；And
Based on described sequencing result, determine the sequence information of described fragmentation DNA target area.
Inventor finds, utilizes the method can effectively determine fragmentation DNA target area sequence information.Further, based on
The Primer composition of the aforementioned present invention of utilization carries out amplification enrichment to the target area of fragmentation DNA, thus notable raising is drawn
The subject range of thing amplification and effective template amount, and it is effectively increased expanding effect specific amplification, and then the enrichment obtained is produced
Thing is after high-flux sequence, and accurately and reliably, the detection sensitivity of fragmentation DNA is high, and repeatable for target area sequencing result
Property is good.Further, the method is particularly suited for the fragmentation DNA detection of trace sample, all anticipates in terms of scientific research and production application
Justice is great, is suitable to promote.
In a fourth aspect of the present invention, the present invention proposes a kind of dress building detection library, fragmentation DNA target area
Put.According to embodiments of the invention, this device utilizes aforesaid Primer composition to expand the target area of fragmentation DNA
Enrichment such that it is able to significantly improve the subject range of primer amplification and effective template amount, it is thus achieved that enriched product i.e. sequencing library
Specificity for target area is good, and then after the high-flux sequence, target area sequencing result accurately and reliably, fragmentation
The detection sensitivity of DNA is high, and favorable repeatability.Further, this device is particularly suited for the target area of fragmentation DNA of trace sample
Territory sequencing library builds.
Referring to Fig. 4, the device 100 that the present invention builds detection library, fragmentation DNA target area is carried out in detail
Explain.
According to embodiments of the invention, this device is provided with foregoing Primer composition, with reference to Fig. 4, described device
100 include: PCR amplification unit the 10, the 2nd PCR amplification unit 20 and a 3rd PCR amplification unit 30.
According to embodiments of the invention, a PCR amplification unit 10 is used for utilizing the first specific primer group and first to lead to
With primer, described fragmentation DNA is carried out a PCR to expand, in order to obtain the first pcr amplification product；2nd PCR amplification unit
20 are connected with a PCR amplification unit 10, are used for utilizing the second specific primer group and the first universal primer to a described PCR
Amplified production carries out the 2nd PCR amplification, in order to obtain the second pcr amplification product；3rd PCR amplification unit 30 and the 2nd PCR expands
Increase unit 20 to be connected, be used for utilizing the first universal primer and the second universal primer that described second pcr amplification product is carried out the 3rd
PCR expands, in order to obtain the 3rd pcr amplification product, and described 3rd pcr amplification product constitutes the target area of described fragmentation DNA
Detection library, territory.
According to embodiments of the invention, farther including pretreatment unit, described pretreatment unit expands with a described PCR
Increase unit to be connected, for, before carrying out a described PCR amplification, described fragmentation DNA carrying out end reparation, 3 ' ends successively
Add poly adenine tail and joint connects.
According to embodiments of the invention, farther include multiple purification unit, for being obtained by aforementioned each unit respectively
Product be purified.Thus, it is thus achieved that Library Quality good, be advantageously used for order-checking detection.
According to embodiments of the invention, a PCR amplification unit 10 is provided with in the 2nd PCR amplification unit 20
AmpliTaq360PCR Master Mix, is used for utilizing AmpliTaq360PCR Master Mix enters respectively
The described PCR amplification of row and described 2nd PCR amplification.
According to embodiments of the invention, a PCR amplification unit 10 is suitable to carry out described first according to following response procedures
PCR expand: 95 DEG C 10 minutes；20 circulations: 95 DEG C 30 seconds, 62 DEG C 30 seconds, 72 DEG C 1 minute；72 DEG C 7 minutes；4 DEG C of preservations.By
This, expanding effect is good.
According to embodiments of the invention, the 2nd PCR amplification unit 20 is suitable to carry out described second according to following response procedures
PCR expand: 95 DEG C 10 minutes；15 circulations: 95 DEG C 30 seconds, 62 DEG C 30 seconds, 72 DEG C 1 minute；72 DEG C 7 minutes；4 DEG C of preservations.By
This, expanding effect is good.
According to embodiments of the invention, the samples sources of fragmentation DNA is not particularly limited.Some according to the present invention are real
Executing example, described fragmentation DNA is selected from plasma dna, urine DNA, perspiration DNA, saliva DNA, seminal fluid DNA, hydrothorax DNA, ascites
At least one DNA, faeces DNA, fossil DNA, paraffin embedding DNA and criminal investigation sample DNA.
According to embodiments of the invention, the target area of described fragmentation DNA comprise selected from point mutation, insert, lack, base
Because of merge sudden change at least one.Thus, the fragmentation DNA target area sequencing library obtained is built, it is possible to be effective to inspection
Survey target area point mutation, insert, lack, gene fusion sudden change.
In a fifth aspect of the present invention, the present invention propose a kind of determine fragmentation DNA target area sequence information be
System 1000.According to embodiments of the invention, with reference to Fig. 5, this system 1000 includes: build fragmentation DNA target area detection literary composition
The device 100 in storehouse, sequencing device 200 and Sequence Determination Means 300.
It is surprisingly found by the inventors that, this system utilizes the Primer composition of the present invention to enter the target area of fragmentation DNA
Row amplification enrichment, thus the notable subject range improving primer amplification and effective template amount, and it is effectively increased expanding effect
Specific amplification, and then the enriched product obtained is after high-flux sequence, can not only effectively determine fragmentation DNA target area
The sequence information in territory, and sequencing result is accurately and reliably, detection sensitivity is high, favorable repeatability.Further, this system is particularly suited for micro-
The fragmentation DNA detection of amount sample, the most significant in terms of scientific research and production application, be suitable to promote.
Referring to Fig. 5, the system 1000 of the determination fragmentation DNA target area sequence information of the present invention is carried out in detail
Explain.
According to embodiments of the invention, build the device 100 in detection library, fragmentation DNA target area, be used for building institute
State the detection library, target area of fragmentation DNA；Sequencing device 200 and the dress building detection library, fragmentation DNA target area
Put 100 to be connected, for being checked order in the detection library, target area of described fragmentation DNA, in order to obtain sequencing result；Sequence
Determine that device 300 is connected with sequencing device 200, for based on described sequencing result, determine described fragmentation DNA target area
Sequence information.
Below in conjunction with embodiment, the solution of the present invention is explained.It will be understood to those of skill in the art that following
Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Unreceipted concrete technology or bar in embodiment
Part, according to the technology described by the document in this area or condition, (such as writing with reference to J. Pehanorm Brooker etc., yellow training hall etc. is translated
" Molecular Cloning: A Laboratory guide ", the third edition, Science Press) or carry out according to product description.Agents useful for same or instrument
Unreceipted production firm person, be can by city available from conventional products, such as can purchase from Illumina company.
Embodiment 1
1. joint design
Prelib-ADT-S:CAAGCAGAAGACGGCATACGAGATIIIIIIIGTGACTGGAGTT CAGACGTGTGCTC
TTCCGATCT (I is index district),
Prelib-ADT-AS:pGATCGGAAGAGC,
Above sequence needs to be annealed into double-strand.
Connection afterproduct structure:
Top:5 '-CAAGCAGAAGACGGCATACGAGATIIIIIIIGTGACTGGAGTTCAGACGTGTGCTC TTCCGA
TCT---AGATCGGAAGAGC-3 ',
Bottom:5 '-CAAGCAGAAGACGGCATACGAGATIIIIIIIGTGACTGGAGTTCAGACGTGTGCTC TTC
CGATCT---AGATCGGAAGAGC-3 ',
The 19 exon designs for EGFR extend specific primer in the same direction.
EGFR19 exon sequence is as follows:
GGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAATTAAGAGAAGCAACATCTCCGAA
AGCCAACAAGGAAATCCTCGAT (SEQ ID NO:11),
EGFR19 exon and upstream and downstream intron region sequence thereof are as follows:
CAGCCCCCAGCAATATCAGCCTTAGGTGCGGCTCCACAGCCCCAGTGTCCCTCACCTTCGGGGTGCATCGCTGGTAA
CATCCACCCAGATCACTGGGCAGCATGTGGCACCATCTCACAATTGCCAGTTAACGTCTTCCTTCTCTCTCTGTCAT
AGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAATTAAGAGAAGCAACATCTCCG
AAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTGCTTTGCTGTGTGGGGGTCCATGGCTCTGAACCTCAGGCCCAC
CTTTTCTCATGTCTGGCAGCTGCTCTGCTCTAGACCCTGCTCATCTCCACATCCTAAATGTTCACTTTCTATGTCTT
TCCCTTTCTAGCTCTAGTGGGTAT (SEQ ID NO:12),
Extend specific primer sequence in the same direction as follows, wherein,
Each specific primer of the first specific primer group is respectively as follows:
N1: TGCCAGTTAACGTCTTCCTTC (SEQ ID NO:3),
N2: ATAGGGACTCTGGATCCCAG (SEQ ID NO:4),
Each specific primer of the second specific primer group is respectively as follows:
M1: GTCTTCCTTCTCTCTCTGTCATAG (SEQ ID NO:5)
M2: TGGATCCCAGAAGGTGAGAAAGTTA (SEQ ID NO:6),
Universal primer sequence is as follows:
First universal primer: CAAGCAGAAGACGGCATACGA (SEQ ID NO:1)；
Second universal primer: AATGATACGGCGACCACCGA (SEQ ID NO:2).
2. take 2ml human normal plasma, extract dissociative DNA.
3. dissociative DNA end is repaired
It is formulated as follows reaction:
Table 1
| Dissociative DNA solution | 75μl |
| T4DNA ligase buffer | 10μl |
| 10mM dNTP mixed liquor | 4μl |
| T4DNA polymerase | 5μl |
| T4DNA phosphorylase | 5μl |
| Klenow enzyme | 1μl |
| Cumulative volume | 100μl |
In PCR instrument, 20 DEG C of temperature are bathed 30 minutes.
120 μ l Ampure XP beads are used to be purified, 32 μ l Elution Buffer eluting.
4.3 ' ends add poly adenine tail
It is formulated as follows reaction:
Table 2
| The DNA solution that end is repaired | 32μl |
| Klenow enzyme buffer liquid | 5μl |
| dATP | 10μl |
| Klenow exo-enzyme | 3μl |
| Cumulative volume | 50μl |
In PCR instrument, 37 DEG C of temperature are bathed 30 minutes.
60 μ l Ampure XP beads are used to be purified, 10 μ l Elution Buffer eluting.
5. jointing
It is formulated as follows reaction:
Table 3
In PCR instrument, 20 DEG C of temperature are bathed 15 minutes.
60 μ l Ampure XP beads are used to be purified, 22 μ l Elution Buffer eluting.
6. target area preenrichment (the i.e. the oneth PCR amplification)
It is formulated as follows reaction:
Table 4
| The DNA solution of jointing | 22μl |
| Amplitaq Gold360 Master Mix | 25μl |
| First specific primer group the+the first universal primer | 2μl |
| GC-enhancer | 1μl |
| Cumulative volume | 50μl |
PCR program is as follows:
A) 95 DEG C 10 minutes；
B) 20 cyclic programs are as follows:
95 DEG C 30 seconds
62 DEG C 30 seconds
72 DEG C 1 minute
C) 72 DEG C 7 minutes
D) 4 DEG C of preservations.
60 μ l Ampure XP beads are used to be purified, 22 μ l Elution Buffer eluting.
7. target area specific enrichment (the i.e. the 2nd PCR amplification)
It is formulated as follows reaction:
Table 5
| The DNA solution of the preenrichment that upper step obtains | 22μl |
| Amplitaq Gold360 Master Mix | 25μl |
| Second specific primer group the+the first universal primer | 2μl |
| GC-enhancer | 1μl |
| Cumulative volume | 50μl |
PCR program is as follows:
A) 95 DEG C 10 minutes；
B) 15 cyclic programs are as follows:
95 DEG C 30 seconds
62 DEG C 30 seconds
72 DEG C 1 minute
C) 72 DEG C 7 minutes
D) 4 DEG C of preservations.
60 μ l Ampure XP beads are used to be purified, 20 μ l Elution Buffer eluting.
8. universal primer amplification (the i.e. the 3rd PCR amplification)
It is formulated as follows reaction:
Table 6
| The DNA solution of the specific enrichment that upper step obtains | 20μl |
| HIFI polymerase mixture | 25μl |
| First universal primer the+the second universal primer | 5μl |
| Cumulative volume | 50μl |
PCR program is as follows:
E) 98 DEG C 45 seconds；
F) 10 cyclic programs are as follows:
98 DEG C 15 seconds
60 DEG C 30 seconds
72 DEG C 30 seconds
G) 72 DEG C 1 minute
H) 4 DEG C of preservations.
60 μ l Ampure XP beads are used to be purified, 30 μ l Elution Buffer eluting.
Taking wherein 5 μ l purified products and carry out 2% agarose gel electrophoresis detection, result is shown in Fig. 6.
Final library, after quantitative fluorescent PCR Quality Control, carries out Illumina company NextSeq500 and carries out 75bp both-end
Order-checking.
By machine data under high-flux sequence after Quality Control is filtered, carry out BWA comparison, for assessing the specificity in library,
Analysis result is shown in Table 7.
Table 7
Result shows, technical scheme successfully can carry out specificity inspection to the target area of fragmentation DNA
Survey.
Embodiment 2
1. joint design
Prelib-ADT-S:CAAGCAGAAGACGGCATACGAGATIIIIIIIGTGACTGGAGTT CAGACGTGTGCTC
TTCCGATCT (I is index district),
Prelib-ADT-AS:pGATCGGAAGAGC,
Above sequence needs to be annealed into double-strand.
Connection afterproduct structure:
Top:5 '-CAAGCAGAAGACGGCATACGAGATIIIIIIIGTGACTGGAGTTCAGACGTGTGCTC TTCCGA
TCT---AGATCGGAAGAGC-3 ',
Bottom:5 '-CAAGCAGAAGACGGCATACGAGATIIIIIIIGTGACTGGAGTTCAGACGTGTGCTC TTC
CGATCT---AGATCGGAAGAGC-3 ',
The 20 exon T790M point mutation designs for EGFR extend specific primer in the same direction.
EGFR20 exon sequence is as follows:
GAAGCCTACGTGATGGCCAGCGTGGACAACCCCCACGTGTGCCGCCTGCTGGGCATCTGCCTCACCTCCACCGTGCA
GCTCATCACGCAGCTCATGCCCTTCGGCTGCCTCCTGGACTATGTCCGGGAACACAAAGACAATATTGGCTCCCAGT
ACCTGCTCAACTGGTGTGTGCAGATCGCAAAG (SEQ ID NO:13),
EGFR20 exon and upstream and downstream intron region sequence thereof are as follows:
GCTAGGTCTTTTGCAGGCACAGCTTTTCCTCCATGAGTACGTATTTTGAAACTCAAGATCGCATTCATGCGTCTTCA
CCTGGAAGGGGTCCATGTGCCCCTCCTTCTGGCCACCATGCGAAGCCACACTGACGTGCCTCTCCCTCCCTCCAGGA
AGCCTACGTGATGGCCAGCGTGGACAACCCCCACGTGTGCCGCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAGC
TCATCACGCAGCTCATGCCCTTCGGCTGCCTCCTGGACTATGTCCGGGAACACAAAGACAATATTGGCTCCCAGTAC
CTGCTCAACTGGTGTGTGCAGATCGCAAAGGTAATCAGGGAAGGGAGATACGGGGAGGGGAGATAAGGAGCCAGGAT
CCTCACATGCGGTCTGCGCTCCTGGGATAGCAAGAGTTTGCCATGGGGATATGTGTGTGCGTGCATGCAGCA(SEQ
ID NO:14),
Extend specific primer sequence in the same direction as follows, wherein,
Each specific primer of the first specific primer group is respectively as follows:
N1: CCAGGAAGCCTACGTGATGGC (SEQ ID NO:7),
N2: TGGGCATCTGCCTCACCTCC (SEQ ID NO:8),
Each specific primer of the second specific primer group is respectively as follows:
M1: GATGGCCAGCGTGGACAACCCCCA (SEQ ID NO:9)
M2: CCTCACCTCCACCGTGCAGCTCAT (SEQ ID NO:10),
Universal primer sequence is as follows:
First universal primer: CAAGCAGAAGACGGCATACGA (SEQ ID NO:1)；
Second universal primer: AATGATACGGCGACCACCGA (SEQ ID NO:2).
2. sample collection
The present embodiment uses 3 samples, and wherein 2 sample (E20-1, E20-2) numeral microdroplet PCR detect in target
Point mutation T790M sudden change (" T790M sudden change " expression: 20 exon the 790th aminoacid of EGFR of different proportion is contained in region
Site is sported methionine Met by threonine Thr), mutation frequency is respectively 6.91% and 0.087%；1 sample (E20-
3) in target area be wild type, i.e. mutation frequency be 0.
For each sample, respectively taking 4ml blood plasma, and extract dissociative DNA 2 parts respectively, a copy of it is standby, for matched group
Experiment.
3. dissociative DNA end is repaired
It is formulated as follows reaction:
Table 8
| Dissociative DNA solution | 75μl |
| T4DNA ligase buffer | 10μl |
| 10mM dNTP mixed liquor | 4μl |
| T4DNA polymerase | 5μl |
| T4DNA phosphorylase | 5μl |
| Klenow enzyme | 1μl |
| Cumulative volume | 100μl |
In PCR instrument, 20 DEG C of temperature are bathed 30 minutes.
120 μ l Ampure XP beads are used to be purified, 32 μ l Elution Buffer eluting.
4.3 ' ends add poly adenine tail
It is formulated as follows reaction:
Table 9
| The DNA solution that end is repaired | 32μl |
| Klenow enzyme buffer liquid | 5μl |
| dATP | 10μl |
| Klenow exo-enzyme | 3μl |
| Cumulative volume | 50μl |
In PCR instrument, 37 DEG C of temperature are bathed 30 minutes.
60 μ l Ampure XP beads are used to be purified, 10 μ l Elution Buffer eluting.
5. jointing
It is formulated as follows reaction:
Table 10
| End is repaired, the 3 ' DNA solutions adding A | 10μl |
| T4 DNA ligase buffer | 25μl |
| 2 μMs of DNA joints | 10μl |
| T4 DNA ligase | 5μl |
| Cumulative volume | 50μl |
In PCR instrument, 20 DEG C of temperature are bathed 15 minutes.
60 μ l Ampure XP beads are used to be purified, 22 μ l Elution Buffer eluting.
6. target area preenrichment (the i.e. the oneth PCR amplification)
It is formulated as follows reaction:
Table 11
| The DNA solution of jointing | 22μl |
| Amplitaq Gold360 Master Mix | 25μl |
| First specific primer group the+the first universal primer | 2μl |
| GC-enhancer | 1μl |
| Cumulative volume | 50μl |
PCR program is as follows:
E) 95 DEG C 10 minutes；
F) 20 cyclic programs are as follows:
95 DEG C 30 seconds
62 DEG C 30 seconds
72 DEG C 1 minute
G) 72 DEG C 7 minutes
H) 4 DEG C of preservations.
60 μ l Ampure XP beads are used to be purified, 22 μ l Elution Buffer eluting.
7. target area specific enrichment (the i.e. the 2nd PCR amplification)
It is formulated as follows reaction:
Table 12
PCR program is as follows:
I) 95 DEG C 10 minutes；
J) 15 cyclic programs are as follows:
95 DEG C 30 seconds
62 DEG C 30 seconds
72 DEG C 1 minute
K) 72 DEG C 7 minutes
L) 4 DEG C of preservations.
60 μ l Ampure XP beads are used to be purified, 20 μ l Elution Buffer eluting.
8. universal primer amplification (the i.e. the 3rd PCR amplification)
It is formulated as follows reaction:
Table 13
| The DNA solution of the specific enrichment that upper step obtains | 20μl |
| HIFI polymerase mixture | 25μl |
| First universal primer the+the second universal primer | 5μl |
| Cumulative volume | 50μl |
PCR program is as follows:
M) 98 DEG C 45 seconds；
N) 10 cyclic programs are as follows:
98 DEG C 15 seconds
60 DEG C 30 seconds
72 DEG C 30 seconds
O) 72 DEG C 1 minute
P) 4 DEG C of preservations.
60 μ l Ampure XP beads are used to be purified, 30 μ l Elution Buffer eluting.
Taking wherein 5 μ l purified products and carry out 2% agarose gel electrophoresis detection, result is shown in Fig. 7.
Final library, after quantitative fluorescent PCR Quality Control, carries out Illumina company NextSeq500 and carries out 75bp both-end
Order-checking.
By machine data under high-flux sequence after Quality Control is filtered, carry out BWA comparison, for assessing the specificity in library,
Analysis result is shown in Table 14.
Table 14
Being further analyzed target area data, the susceptiveness of assessment T790M abrupt climatic change, analysis result is shown in Table
15。
Table 15
9. matched group experiment ARMS method detection
Wherein matched group uses the EGFR genetic mutation detectable of Beijing Xinnuo Meidi Gene Inspection Technology Co., Ltd.
Box (PCR-fluorescence probe method), article No.: SMD-02-041, the sudden change to the T790M of another part of plasma dna solution of 3 samples
Situation detects.Testing result is shown in Table 16.
Table 16
| Sample | △ Ct value |
| E20-1 | △ Ct=8 |
| E20-2 | △ Ct=12 |
| E20-3 | Wild type |
In the result of table 16, △ Ct≤8 are defined as saltant type, and sudden change content is 1%-100%；△ Ct > 8 is defined as wild
Type, or sudden change is less than 1%.
Result shows, technical scheme is compared at present conventional DNA mutation detection method and technology (such as
ARMS method) there is the higher advantage of sensitivity, in the case of ARMS method is merely capable of the detection sudden change higher than 1%, the present invention
Technical scheme can detect the fragmentation DNA of mutation frequency as little as even below 0.1%.
In the description of this specification, reference term " embodiment ", " some embodiments ", " example ", " specifically show
Example " or the description of " some examples " etc. means to combine this embodiment or example describes specific features, structure, material or spy
Point is contained at least one embodiment or the example of the present invention.In this manual, to the schematic representation of above-mentioned term not
Necessarily refer to identical embodiment or example.And, the specific features of description, structure, material or feature can be any
One or more embodiments or example in combine in an appropriate manner.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that: not
These embodiments can be carried out multiple change in the case of departing from the principle of the present invention and objective, revise, replace and modification, this
The scope of invention is limited by claim and equivalent thereof.
Claims (10)
1. the Primer composition being used for detecting fragmentation DNA target area, it is characterised in that including:
First primer sets, described first primer sets comprises the first universal primer and the second universal primer, described first universal primer
With the two ends that the target spot of described second universal primer is positioned at described target area, it is respectively used to constitute the 5 ' ends and 3 ' of sequencing library
End；And
Second primer sets, described second primer sets includes the first specific primer group and the second specific primer group,
Wherein,
Described first specific primer group and described second specific primer group all comprise n bar target region-specific primers, and
Described first specific primer group and described second specific primer group must are fulfilled for following condition:
(1) each specific primer of described first specific primer group and described second specific primer group extends the most in the same direction, target
Point is respectively positioned on 5 ' ends or the 3 ' ends of described fragmentation DNA target area；
(2) arbitrary integer of n=1-5, as a example by n=5, if each specific primer of described first specific primer group is respectively
For: N1、N2、N3、N4、N5, each specific primer of described second specific primer group is respectively as follows: M1、M2、M3、M4、M5, with special
Distance between target spot and the target area of property primer is criterion, described first specific primer group and described second special
Distance relation between each specific primer of property primer sets is:
N1>N2>N3>N4>N5；
M1>M2>M3>M4>M5；
N1>M1；
N2>M2；
N3>M3；
N4>M4；And
N5>M5,
Wherein, the target spot of each specific primer of described first specific primer group and described second specific primer group, with institute
The target spot stating the first universal primer lays respectively at the two ends of described target area, is positioned at institute with the target spot of described second universal primer
State the same end of target area.
Primer sets the most according to claim 1, it is characterised in that for each specific primer group, it is each that it comprises
Specific primer is spaced 0-50 to base pair successively based on the order on genomic locations,
Optionally, N1With M1Corresponding genomic locations has overlap, N2With M2Corresponding genomic locations has overlap, N3 and M3Corresponding
Genomic locations have overlap, N4With M4Corresponding genomic locations has overlap, N5With M5Corresponding genomic locations has overlap,
Preferably overlapping 0-20 base pair,
Optionally, the G/C content of each specific primer is 30%-70%, Tm value and is 55-68 DEG C.
Primer sets the most according to claim 1, it is characterised in that two universal primers are respectively as follows:
First universal primer: CAAGCAGAAGACGGCATACGA (SEQ ID NO:1)；
Second universal primer: AATGATACGGCGACCACCGA (SEQ ID NO:2),
Optionally, described fragmentation DNA target area is EGFR gene 19 exon, and n=2, and described first specificity draws
Each specific primer of thing group is respectively as follows:
N1: TGCCAGTTAACGTCTTCCTTC (SEQ ID NO:3),
N2: ATAGGGACTCTGGATCCCAG (SEQ ID NO:4)；And
Each specific primer of described second specific primer group is respectively as follows:
M1: GTCTTCCTTCTCTCTCTGTCATAG (SEQ ID NO:5)
M2: TGGATCCCAGAAGGTGAGAAAGTTA (SEQ ID NO:6),
Optionally, described fragmentation DNA target area is EGFR gene 20 exon, and n=2, and described first specificity draws
Each specific primer of thing group is respectively as follows:
N1: CCAGGAAGCCTACGTGATGGC (SEQ ID NO:7),
N2: TGGGCATCTGCCTCACCTCC (SEQ ID NO:8)；And
Each specific primer of described second specific primer group is respectively as follows:
M1: GATGGCCAGCGTGGACAACCCCCA (SEQ ID NO:9)
M2: CCTCACCTCCACCGTGCAGCTCAT (SEQ ID NO:10),
Optionally, described fragmentation DNA is selected from plasma dna, urine DNA, perspiration DNA, saliva DNA, seminal fluid DNA, hydrothorax
At least one DNA, ascites DNA, faeces DNA, fossil DNA, paraffin embedding DNA and criminal investigation sample DNA,
Optionally, the target area of described fragmentation DNA comprise selected from point mutation, insert, lack, gene fusion is suddenlyd change at least
One of.
4. the method in the detection library, target area building fragmentation DNA, it is characterised in that
Utilize the Primer composition described in any one of claim 1-3, be enriched with described fragmentation DNA target area by PCR amplification
The sequence in territory.
Method the most according to claim 4, it is characterised in that described method includes:
Utilize the first specific primer group and the first universal primer that described fragmentation DNA carries out a PCR amplification, in order to obtain
First pcr amplification product；
Utilize the second specific primer group and the first universal primer that described first pcr amplification product carries out the 2nd PCR amplification, with
Just the second pcr amplification product is obtained；And
Utilize the first universal primer and the second universal primer that described second pcr amplification product carries out the 3rd PCR amplification, in order to obtain
Obtaining the 3rd pcr amplification product, described 3rd pcr amplification product constitutes the detection library, target area of described fragmentation DNA,
Optionally, before carrying out a described PCR amplification, farther include:
Described fragmentation DNA carries out end reparation successively, 3 ' ends add poly adenine tail and joint connects,
Optionally, farther include to be purified the product of each step,
Optionally, AmpliTaq is utilized360PCR Master Mix carries out described PCR amplification and described 2nd PCR
Amplification,
Optionally, the response procedures of a described PCR amplification is:
95 DEG C 10 minutes；
20 circulations: 95 DEG C 30 seconds, 62 DEG C 30 seconds, 72 DEG C 1 minute；
72 DEG C 7 minutes；
4 DEG C of preservations,
Optionally, the response procedures of described 2nd PCR amplification is:
95 DEG C 10 minutes；
15 circulations: 95 DEG C 30 seconds, 62 DEG C 30 seconds, 72 DEG C 1 minute；
72 DEG C 7 minutes；
4 DEG C of preservations.
Method the most according to claim 4, it is characterised in that described fragmentation DNA be selected from plasma dna, urine DNA,
Perspiration DNA, saliva DNA, seminal fluid DNA, hydrothorax DNA, ascites DNA, faeces DNA, fossil DNA, paraffin embedding DNA and criminal investigation sample
At least one DNA,
Optionally, the target area of described fragmentation DNA comprise selected from point mutation, insert, lack, gene fusion is suddenlyd change at least
One of.
7. the method determining fragmentation DNA target area sequence information, it is characterised in that including:
According to the method described in any one of claim 4-6, build the detection library, target area of described fragmentation DNA；
Is checked order in the detection library, target area of described fragmentation DNA, in order to obtain sequencing result；And
Based on described sequencing result, determine the sequence information of described fragmentation DNA target area.
8. the device building detection library, fragmentation DNA target area, it is characterised in that be provided with claim 1-3 and appoint
One described Primer composition, described device includes:
Oneth PCR amplification unit, a described PCR amplification unit is used for utilizing the first specific primer group and the first universal primer
Described fragmentation DNA is carried out a PCR amplification, in order to obtain the first pcr amplification product；
2nd PCR amplification unit, described 2nd PCR amplification unit is connected with a described PCR amplification unit, is used for utilizing second
Specific primer group and the first universal primer carry out the 2nd PCR amplification to described first pcr amplification product, in order to obtain second
Pcr amplification product；And
3rd PCR amplification unit, described 3rd PCR amplification unit is connected with described 2nd PCR amplification unit, is used for utilizing first
Universal primer and the second universal primer carry out the 3rd PCR amplification to described second pcr amplification product, in order to obtain the 3rd PCR and expand
Volume increase thing, described 3rd pcr amplification product constitutes the detection library, target area of described fragmentation DNA.
Device the most according to claim 8, it is characterised in that farther include pretreatment unit, described pretreatment unit
It is connected with a described PCR amplification unit, for, before carrying out a described PCR amplification, described fragmentation DNA being entered successively
Row end is repaired, 3 ' ends add poly adenine tail and joint connects,
Optionally, farther include multiple purification unit, for respectively the product that each unit obtains being purified,
Optionally, a described PCR amplification unit and described 2nd PCR amplification unit are provided with AmpliTaq
360PCR Master Mix, is used for utilizing AmpliTaq360PCR Master Mix carries out a described PCR respectively
Amplification and described 2nd PCR amplification,
Optionally, a described PCR amplification unit be suitable to according to following response procedures carry out described oneth PCR amplification:
95 DEG C 10 minutes；
20 circulations: 95 DEG C 30 seconds, 62 DEG C 30 seconds, 72 DEG C 1 minute；
72 DEG C 7 minutes；
4 DEG C of preservations,
Optionally, described 2nd PCR amplification unit be suitable to according to following response procedures carry out described 2nd PCR amplification:
95 DEG C 10 minutes；
15 circulations: 95 DEG C 30 seconds, 62 DEG C 30 seconds, 72 DEG C 1 minute；
72 DEG C 7 minutes；
4 DEG C of preservations,
Optionally, described fragmentation DNA is selected from plasma dna, urine DNA, perspiration DNA, saliva DNA, seminal fluid DNA, hydrothorax
At least one DNA, ascites DNA, faeces DNA, fossil DNA, paraffin embedding DNA and criminal investigation sample DNA,
Optionally, the target area of described fragmentation DNA comprise selected from point mutation, insert, lack, gene fusion is suddenlyd change at least
One of.
10. the system determining fragmentation DNA target area sequence information, it is characterised in that including:
The device in the detection library, structure fragmentation DNA target area described in claim 8 or 9, is used for building described fragmentation
The detection library, target area of DNA；
Sequencing device, described sequencing device is connected, for right with the device in detection library, described structure fragmentation DNA target area
Checking order in the detection library, target area of described fragmentation DNA, in order to obtains sequencing result；And
Sequence Determination Means, described Sequence Determination Means is connected with described sequencing device, for based on described sequencing result, determines
The sequence information of described fragmentation DNA target area.
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| CN106834286A (en) * | 2017-04-05 | 2017-06-13 | 北京泛生子基因科技有限公司 | The primer combination of one-step method rapid build amplification sublibrary |
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