CN109337973A - A kind of primer design method, primer, kit, method and system copying number variation for verifying specific DNA fragments - Google Patents
A kind of primer design method, primer, kit, method and system copying number variation for verifying specific DNA fragments Download PDFInfo
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Abstract
The present invention provides a kind of for verifying the primer design method of specific DNA fragments copy number variation, the present invention also provides a kind of for verifying primer, the method and system of specific DNA fragments copy number variation, compared with prior art, the region of design primer increases in primer design method of the present invention, it is no longer limited to 80bp to 150bp, design of primers difficulty greatly reduces.Sequencing result is very intuitive in verification method of the present invention, more convenient to the judgement of deletion mutation.It can influence to avoid homology to verifying using primer design method of the invention, verifying primer, verification method and verifying system；It can not only determine that CNV whether there is using verification method of the invention and verifying system, can also determine whether CNV and Minor variations are on same chromosome.
Description
Technical field
The present invention relates to a kind of for verifying primer design method, primer, the reagent of specific DNA fragments copy number variation
Box, method and system.
Background technique
Human chromosomal has 46, and wherein autosome has 22 pairs, occurs in pairs, and in each pair of chromosome, one is come
From in paternal, a maternal is called homologue.Chromosome is mainly made of DNA and protein, wherein DNA, that is, deoxidation core
Ribosomal ribonucleic acid sequence (also known as nucleotide sequence or base sequence) decides hereditary information.DNA is a kind of long-chain polymer, composition
Unit is four kinds of deoxynucleotides, i.e. adenyl-deoxyribonucleotide (dAMP), thymidylic acid (dTMP), cytimidine
Deoxynucleotide (dCMP), guanine deoxyribonucleoside are sour (dGMP).Determine diversity of organism is exactly four in deoxynucleotide
Kind base, adenine (adenine, A), thymidine (thymine, T), cytimidine (cytosine, C) and guanine
(guanine, G), DNA sequence dna usually indicate with the sequence of tetra- kinds of bases of ATCG, referred to as base sequence or nucleotide sequence, A
It is matched with T, C and G are matched, and forward direction refers to 5 ' ends to 3 ' extreme directions.
Genetic mutation refers to heritable variation that genomic DNA molecule occurs, and is related to clip size according to variation, can divide
For Minor variations and number variation is copied, wherein copy number variation (Copy number variations, CNVs) refers to that length is big
It makes a variation in the missing of the DNA fragmentation of 1kb or repetition.There are many ways to CNVs is detected, such as chromosome microarray chip technology
(chromosomal microarray, CMA), mononucleotide genotyping technique, multiplex ligation-dependent probe amplification
(Multiplex ligation-dependent probe amplification, MLPA) and at present by clinical research
Widely applied high throughput sequencing technologies.
It for the CNVs detected as CMA, high-flux sequence etc., generally requires further to be verified with other methods, commonly use
Method have MLPA, sxemiquantitative qPCR etc..Wherein qPCR is one of most common method, and still, qPCR amplification length requires tight
Lattice, usually 80bp to 150bp, when target fragment is there are when higher homology, design of primers is difficult and is easy to failure, into
And authentication failed.And MLPA is the finished product reagent of Reagent Company's exploitation, there is no correspondent probes for big polygenes.One of situation
Be detect a heterozygosis Minor variations, meanwhile, nearby again detect a heterozygous deletion CNV, at this point, homology is very high
When, often it is difficult to verify the existence of CNV by qPCR.
Summary of the invention
It is an object of that present invention to provide a kind of for verifying the primer design method of specific DNA fragments copy number variation, base
Should primer design method, the present invention also provides it is a kind of for verify specific DNA fragments copy number variation primer, reagent
Box, method and system, to solve the problems mentioned in the above background technology.
To achieve the above object, the technical solution taken: a kind of for verifying drawing for specific DNA fragments copy number variation
Object design method, the primer design method the following steps are included:
For it is known there are heterozygosis Minor variations and there may be heterozygous deletion copy number variation specific DNA to be verified
Segment, design is at least a pair of for detecting the primer of the heterozygosis Minor variations, wherein a primer in the primer pair designed
Positioned at the upstream or downstream of Minor variations, another primer is located at missing copy number variable region to be verified, the primer of design
To the primer to copy number variation for validating DNA segment.
Heterozygosis Minor variations refer to that there are Minor variations on the item chromosome in homologue, and another is dyed
The Minor variations are not present on body corresponding position.Heterozygous deletion copy number variation refers to the item chromosome in homologue
It is upper to there is missing copy number variation, and there is no the missings to copy number variation on another item chromosome corresponding position.
The primer of the DNA of design is a bit of single stranded DNA, and upstream primer (i.e. forward primer) is that (have with DNA normal chain
Adopted chain) it is complementary, downstream primer (i.e. reverse primer) be it is consistent with DNA normal chain (i.e. sense strand), design of primers generally requires
Primer length is 15 to 30 bases, and primer cannot have primer dimer and hairpin structure, and primer will have specificity i.e. DNA sequence dna
Conserved region etc..In the present invention, the method for specific DNA fragments design primer to be verified is, in the primer pair of design
One primer is located at the upstream or downstream of Minor variations, and another primer is located at the region CNV to be verified.Specifically, work as CNV
When region is located at Minor variations upstream, a primer in the primer pair of design is located at the downstream of Minor variations, another primer
Positioned at the region CNV to be verified；When the region CNV is located at Minor variations downstream, a primer in the primer pair of design is located at micro-
The upstream of small variation, another primer are located at the region CNV to be verified；The one of benefit designed in this way be can expand arrive it is small
Variation achievees the purpose that detect Minor variations.
Preferably, Minor variations are less than at a distance from missing copy number variable region in the specific DNA fragments to be verified
800bp。
Present invention specific DNA fragments to be verified are to be detected by the prior art there are heterozygosis Minor variations and may be deposited
In the DNA fragmentation of heterozygous deletion copy number variation.Generally, the specific DNA segment to be verified is examined through high-flux sequence
It measures there are heterozygosis Minor variations and there may be the DNA fragmentations of heterozygous deletion copy number variation.
Preferably, the primer pair of the design be two pairs, respectively the Outside primer of nested PCR amplification to and inner primer
It is right.Nested PCR Technique is merged in primer design method of the present invention can influence to avoid homology to verifying.
Preferably, the specific DNA fragments to be verified are CYP11B1 gene, and heterozygosis Minor variations are located at CYP11B1 base
C.1391_1393dup because of the 8th exon, there may be the changes of heterozygous deletion copy number for the heterozygosis Minor variations upstream vicinity
It is different.
The present invention provides a kind of for verifying the primer of specific DNA fragments copy number variation, and the primer is using upper
State what the primer design method designed.
Preferably, the primer by nested PCR amplification Outside primer to and inner primer to forming:
Outside primer pair:
Upstream primer F1:5 '-TACTTGGGATTGTGATGTG-3 ' (SEQ ID NO:1),
Downstream primer R1:5 '-AAACCACAGCACCCTTGCATGGCCA-3 ' (SEQ ID NO:2)；
Inner primer pair:
Upstream primer F2:5 '-TAAACAGCGTCACCCAGCAG-3 ' (SEQ ID NO:3),
Downstream primer R2:5 '-CTTAGCCTGGCAAACCCTGG-3 ' (SEQ ID NO:4).
The present invention provides a kind of for verifying the kit of specific DNA fragments copy number variation, and the kit includes
Primer described above.
The present invention provides a kind of methods for verifying specific DNA fragments copy number variation, and the method includes as follows
Step:
(1) to include known there are heterozygosis Minor variations and there may be the to be verified specific of heterozygous deletion copy number variation
The DNA sample of DNA fragmentation is template, is expanded, will be expanded using the primer that primer design method described above designs
Increase obtained product and carries out sanger sequencing；
(2) the sanger sequencing result that step (1) obtains is compared with normal controls sequence；Knot is verified after comparison
Fruit is as follows:
If sanger sequencing result shows that the Minor variations in the specific DNA fragments to be verified are homozygous Minor variations,
Then there are heterozygous deletions to copy number variation for the specific DNA fragments to be verified, and is located at different homologous dyeing from Minor variations
On body；
If sanger sequencing result shows that the Minor variations in the specific DNA fragments to be verified are heterozygosis Minor variations,
Then there is no missing copy number variations for the specific DNA fragments to be verified；
If sanger sequencing result is shown in the specific DNA fragments to be verified and does not measure Minor variations, this is to be tested
Demonstrate,proving specific DNA fragments, there are heterozygous deletions to copy number variation, and is located on same homologue with Minor variations.
Sanger method is that the point according to nucleotide in a certain fixation starts, and is terminated at some specific base at random,
And it generates in each base followed by fluorescent marker with a series of nucleotide of A, T, C, G four groups of different lengths terminated,
Then electrophoresis is detected in urea-denatured PAGE glue, to obtain a kind of method of visible DNA base sequence.
Preferably, the homology of other DNA sequence dnas is greater than etc. in the specific DNA fragments to be verified and the DNA sample
In 80%.It is highly preferred that the homology of other DNA sequence dnas is greater than etc. in the specific DNA fragments to be verified and the DNA sample
In 90%.When the homology of other DNA sequence dnas in the specific DNA fragments to be verified and the DNA sample is more than or equal to 80%
When, it is difficult to design suitable primer using qPCR, especially when in the specific DNA fragments to be verified and the DNA sample
When the homology of other DNA sequences is more than or equal to 90%, it more difficult to design suitable qPCR primer.
The system that the present invention provides a kind of to copy number variation for verifying specific DNA fragments, the system comprises
Gene amplification device, the gene amplification device include known there are heterozygosis Minor variations and possible for expanding
There are the DNA sample of the specific DNA fragments to be verified of heterozygous deletion copy number variation, the primer used that expands is using upper
State the primer that the primer design method designs；
Gene sequencing device, the gene sequencing device are connected with the gene amplification device, and for the expansion
The product of increasing carries out sanger sequencing, to obtain the sanger sequencing sequence of Minor variations in specific DNA fragments to be verified；
Comparison device, the comparison device are connected with the gene order-checking device, prestore just in the comparison device
Ordinary person's reference sequences information, the sanger sequencing sequence and normal person's reference sequences that the comparison device is used to obtain are believed
Breath is compared, and the Minor variations situation determined by sanger sequencing result is obtained based on comparison result, then in conjunction with to
There are this Given informations of heterozygosis Minor variations in verifying specific DNA fragments lacks to verify heterozygosis in specific DNA fragments to be verified
The case where losing copy number variation；If sanger sequencing result shows that the Minor variations in the specific DNA fragments to be verified are pure
Minor variations are closed, then there are heterozygous deletions to copy number variation for the specific DNA fragments to be verified, and from Minor variations positioned at different
Homologue on；If sanger sequencing result shows that the Minor variations in the specific DNA fragments to be verified are that heterozygosis is micro-
Small variation, then there is no missing copy number variations for the specific DNA fragments to be verified；If sanger sequencing result be shown in it is described to
Not measuring Minor variations in verifying specific DNA fragments, then there are heterozygous deletions to copy number variation for the specific DNA fragments to be verified,
And it is located on same homologue with Minor variations；And
As a result output device, the result output device is connected with the comparison device, to export verification result.
The utility model has the advantages that the primer that the present invention provides a kind of for verifying the primer of specific DNA fragments copy number variation is set
Meter method, the present invention also provides a kind of for verifying primer, the method and system of specific DNA fragments copy number variation, compares
The prior art, the region of design primer increases in primer design method of the present invention, is no longer limited to 80bp to 150bp, primer is set
Meter difficulty greatly reduces.Sequencing result is very intuitive in verification method of the present invention, more convenient to the judgement of deletion mutation.Using
Primer design method of the invention, verifying primer, verification method and verifying system can influences to avoid homology to verifying；It adopts
It can not only determine that CNV whether there is with verification method of the invention and verifying system, can also determine that CNV is with Minor variations
It is no to be on same chromosome.
Detailed description of the invention
Fig. 1 is the schematic diagram for copying number variation design of primers in the embodiment of the present invention 1 for specific DNA fragments, in figure: 1,
Upstream primer F2；2, homologue A；3, homologue B；4, absent region to be verified；5, heterozygosis Minor variations；6, under
Swim primer R2；
Fig. 2 is 1 middle and upper reaches primers F of the embodiment of the present invention, 2 sequencing result map, in figure: 1, normal person's reference sequences；2, sample
This sequence；3, Minor variations are c.1391_1393dup；
Fig. 3 is 1 middle and lower reaches primer R2 sequencing result map of the embodiment of the present invention, in figure: 1, normal person's reference sequences；2, sample
This sequence；3, Minor variations are c.1391_1393dup.
Specific embodiment
To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with specific embodiment to the present invention
It is described further.
Embodiment 1
We detect a heterozygosis Minor variations CYP11B1 gene (with reference to transcript NM_ by high-flux sequence
000497.3) the 8th exon c.1391_1393dup, be located at homologue A on and one section of nucleotide sequence of upstream vicinity
Heterozygous deletion, range of loss are about about 37kb, and position is chr8:143956640-143994311, this segment includes CYP11B1
Next gene 1-7 exon is verified for the heterozygous deletion segment.Attempt use qPCR design primer, discovery by
In CYP11B1 gene there are pseudogene CYP11B2, homology is very high, and up to 90% or more, and qPCR requires high, amplification length one
As require in 80bp to 150bp, be difficult to design suitable primer, we once attempted absent region to be detected design it is several right
QPCR primer；Such as: one of primer: upstream primer 5 '-TGGCAACAGGAGAGACAGGAT-3 ' (SEQ ID NO:5), downstream is drawn
Object: 5 '-GGCTCCGTATCAACCAGAGAAA-3 ' (SEQ ID NO:6), the two of primer: upstream primer 5 '-
TCCACCGTCCAGCTCATGT-3 ' (SEQ ID NO:7), downstream primer: 5 '-GCCTCAAAGTGCTCCTTCCA-3 ' (SEQ
ID NO:8), the three of primer: upstream primer 5 '-AGGACGTGGAGAAGCTGCAA-3 ' (SEQ ID NO:9), downstream primer:
5' -TGCCCACGATGTTGTCTGTAG-3'(SEQ ID NO:10).Because these primers and amplification region are located at, there are homologous sequences
The region of column can have an impact when to region quantitative amplification to be measured, can not correctly judge the state of missing, qPCR the failure of an experiment.
Next, we take method of the invention to verify, process description is as follows:
Firstly, absent region and Minor variations c.1391_1393dup downstream design primer in high-flux sequence detection:
Upstream primer F1:5 '-TACTTGGGATTGTGATGTG-3 ' (SEQ ID NO:1)
Downstream primer R1:5 '-AAACCACAGCACCCTTGCATGGCCA-3 ' (SEQ ID NO:2)
Is carried out by long PCR, obtains product P using the sample DNA of former high-flux sequence detection as template for primer using this.Its
In, reaction system setting such as table 1:
1 first round of table PCR reaction system
| Reagent | Volume (μ l) |
| ddH2O | 12.5 |
| 2X Phusion Green HS II HF Master Mix | 25 |
| Forward primer F1 | 2.5 |
| Reverse primer R1 | 2.5 |
| DNA profiling | 4 |
| DMSO (3%) | 1.5 |
PCR response procedures setting such as table 2:
2 first round of table PCR response procedures
Long PCR is carried out, it can be to avoid the interference of high homology segment such as pseudogene.
Then, c.1391_1393dup downstream and drawing in the absent region of high-flux sequence detection and Minor variations respectively
The amplification section design primer of object F1 and primer R1, schematic diagram is as shown in Figure 1:
Upstream primer F2:5 '-TAAACAGCGTCACCCAGCAG-3 ' (SEQ ID NO:3)
Downstream primer R2:5 '-CTTAGCCTGGCAAACCCTGG-3 ' (SEQ ID NO:4)
Downstream primer R2 is located at the downstream of Minor variations, and wherein upstream primer F2 is located at the region CNV to be verified.
Using this to primer, PCR amplification is carried out by template of long PCR product P, reaction system setting such as table 3:
Table 3 second takes turns PCR reaction system
Reaction condition such as table 4:
Table 4 second takes turns PCR response procedures
| Temperature | Time | Reaction cycle number |
| 98℃ | 3min | 1 |
| 98℃ | 10s | 30 |
| 63℃ | 30s | 1 |
| 72℃ | 1min | 1 |
| 72℃ | 5min | 1 |
| 25℃ | ∞ | 1 |
Then, PCR product carries out sanger sequencing, and sequencing primer is upstream primer F2 and downstream primer R2, obtains such as Fig. 2
With the result of Fig. 3: the sequencing result of upstream primer F2 and downstream primer R2, sample sequence and normal person's reference sequences compare, all
C.1391_1393dup, display Minor variations are homozygous.
Finally, interpretation of result is carried out, it is as follows:
(1) if it is homozygous for measuring Minor variations c.1391_1393dup: speculating CNV (chr8:143956640-
143994311) exist, and be located on different homologues from Minor variations.
(2) if c.1391_1393dup measure Minor variations is heterozygosis: speculating CNV and be not present.
(3) Minor variations are not measured c.1391_1393dup, i.e., only measure wild type: speculating that CNV exists, and with it is small
Variation is located on same homologue.
Result detected by this experiment belongs to the first situation, i.e. c.1391_1393dup Minor variations are homozygous, in conjunction with
High-flux sequence Minor variations c.1391_1393dup heterozygosis, thus it is speculated that another homologous chromosomal segments of Minor variations, that is, homologous
Chromosome B is not amplified out because there is missing, therefore does not have sequencing data, and therefore, which supports high pass
Measure the deletion mutation of sequence detection.It is proved to be successful.
In the embodiment of the present invention, the primers F 1 and primer R1 carry out long PCR amplification can to avoid the influence of homologous sequence,
Homology is not high, when being such as lower than 80%, can ignore this step without long PCR.CNV is verified by sanger PCR sequencing PCR, is had
Effect ground reduces the limitation of PCR primer design, meanwhile, it can speculate whether adjacent Minor variations and CNV are in same dyeing
On body, certain meaning might have for the explanation of autosomal recessive hereditary diseases.
For comparing, qPCR is because of design requirement height, and amplification length is shorter, it is desirable that and stringent, design of primers difficulty is high, and
Stable experiment is relatively poor.And when homology is higher, often it is difficult to design suitable primer, therefore be difficult to be verified
Experiment.Since qPCR is can not to learn the opposite position of absent region and Minor variations to the half-quantitative detection of region to be measured amplification
It sets, i.e., whether on same homologue.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of range is protected, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should
Understand, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the essence of technical solution of the present invention
And range.
Sequence table
<110>Guangzhou Kingmed Center for Clinical Laboratory Co., Ltd., Guangzhou Jin Yu medical test Group Plc
<120>a kind of for verifying primer design method, primer, the kit, side of specific DNA fragments copy number variation
Method and system
<160> 10
<170> PatentIn version 3.3
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Claims (10)
1. a kind of for verifying the primer design method of specific DNA fragments copy number variation, which is characterized in that the design of primers
Method the following steps are included:
For it is known there are heterozygosis Minor variations and there may be heterozygous deletion copy number variation specific DNA fragments to be verified,
Design is at least a pair of for detecting the primer of the heterozygosis Minor variations, wherein a primer in the primer pair designed is positioned at micro-
The upstream or downstream of small variation, another primer are located at missing copy number variable region to be verified, and the primer pair of design is to use
In the primer of verifying specific DNA fragments copy number variation.
2. primer design method according to claim 1, which is characterized in that small in the specific DNA fragments to be verified
Variation is less than 800bp at a distance from missing copy number variable region.
3. primer design method according to claim 1, which is characterized in that the specific DNA fragments to be verified are through height
Flux sequencing is detected there are heterozygosis Minor variations and there may be the DNA fragmentations of heterozygous deletion copy number variation.
4. primer design method according to claim 1, which is characterized in that the primer pair of the design is two pairs, respectively
For nested PCR amplification Outside primer to and inner primer pair.
5. primer design method according to claim 1, which is characterized in that the specific DNA fragments to be verified are
C.1391_1393dup CYP11B1 gene, heterozygosis Minor variations are located at the 8th exon of CYP11B1 gene, the heterozygosis is small
Making a variation, there may be heterozygous deletions to copy number variation for upstream vicinity.
6. a kind of for verifying the primer of specific DNA fragments copy number variation, which is characterized in that the primer is using such as right
It is required that any primer design method of 1-5 designed；
Preferably, the primer by nested PCR amplification Outside primer to and inner primer to forming:
Outside primer pair:
Upstream primer F1:5 '-TACTTGGGATTGTGATGTG-3 ',
Downstream primer R1:5 '-AAACCACAGCACCCTTGCATGGCCA-3 '；
Inner primer pair:
Upstream primer F2:5 '-TAAACAGCGTCACCCAGCAG-3 ',
Downstream primer R2:5 '-CTTAGCCTGGCAAACCCTGG-3 '.
7. a kind of for verifying the kit of specific DNA fragments copy number variation, which is characterized in that including such as claim 6 institute
The primer stated.
8. a kind of method for verifying specific DNA fragments copy number variation, which comprises the steps of:
(1) to include known there are heterozygosis Minor variations and there may be the specific DNA to be verified of heterozygous deletion copy number variation
The DNA sample of segment is template, is carried out using the primer that primer design method a method as claimed in any one of claims 1 to 5 designs
Amplification, the product that amplification is obtained carry out sanger sequencing；
(2) the sanger sequencing result that step (1) obtains is compared with normal controls sequence；Verification result is such as after comparison
Under:
If sanger sequencing result shows that the Minor variations in the specific DNA fragments to be verified, should for homozygous Minor variations
There are heterozygous deletions to copy number variation for specific DNA fragments to be verified, and is located on different homologues from Minor variations；
If sanger sequencing result shows that the Minor variations in the specific DNA fragments to be verified are heterozygosis Minor variations, should
There is no missing copy number variations for specific DNA fragments to be verified；
If sanger sequencing result is shown in the specific DNA fragments to be verified and does not measure Minor variations, the spy to be verified
Determining DNA fragmentation, there are heterozygous deletions to copy number variation, and is located on same homologue with Minor variations.
9. according to the method described in claim 8, it is characterized in that, in the specific DNA fragments to be verified and the DNA sample
The homology of other DNA sequence dnas is more than or equal to 80%；
Preferably, the specific DNA fragments to be verified and the homology of other DNA sequence dnas in the DNA sample are more than or equal to
90%.
10. a kind of system for verifying specific DNA fragments copy number variation, which is characterized in that including
Gene amplification device, the gene amplification device, for expand include it is known there are heterozygosis Minor variations and there may be
Heterozygous deletion copies the DNA sample of the specific DNA fragments to be verified of number variation, and the primer used that expands is using such as right
It is required that the primer that any primer design method of 1-5 designs；
Gene sequencing device, the gene sequencing device are connected with the gene amplification device, and for the amplification
Product carries out sanger sequencing, to obtain the sanger sequencing sequence of Minor variations in specific DNA fragments to be verified；
Comparison device, the comparison device are connected with the gene order-checking device, prestore normal person in the comparison device
Reference sequences information, the comparison device for will obtained sanger sequencing sequence and normal person's reference sequences information into
Row compares, and the Minor variations situation determined by sanger sequencing result is obtained based on comparison result, then in conjunction with to be verified
There are this Given informations of heterozygosis Minor variations in specific DNA fragments copies to verify heterozygous deletion in specific DNA fragments to be verified
The case where shellfish number variation；If sanger sequencing result shows that the Minor variations in the specific DNA fragments to be verified are homozygous micro-
Small variation, then there are heterozygous deletions to copy number variation for the specific DNA fragments to be verified, and is located at from Minor variations different same
On source chromosome；If sanger sequencing result shows that the Minor variations in the specific DNA fragments to be verified are the small change of heterozygosis
Different, then there is no missing copy number variations for the specific DNA fragments to be verified；If sanger sequencing result is shown in described to be verified
Minor variations are not measured in specific DNA fragments, then there are heterozygous deletions to copy number variation for the specific DNA fragments to be verified, and
It is located on same homologue with Minor variations；And
As a result output device, the result output device is connected with the comparison device, to export verification result.
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| CN111304299A (en) * | 2019-12-11 | 2020-06-19 | 北京泛生子基因科技有限公司 | Primer combination, kit and method for detecting copy number variation of autosome |
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