CN108103154A - Judge the method for copy number of target genes in genome - Google Patents
Judge the method for copy number of target genes in genome Download PDFInfo
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- CN108103154A CN108103154A CN201711482313.5A CN201711482313A CN108103154A CN 108103154 A CN108103154 A CN 108103154A CN 201711482313 A CN201711482313 A CN 201711482313A CN 108103154 A CN108103154 A CN 108103154A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Abstract
The invention discloses the method for judging copy number of target genes in genome, by the use of the gene of a known copy number in the genome of species to be measured as reference gene A；A genetic fragment ABm is built, thereon the mutant fragments Bm of the mutant fragments Am of the A sequences containing identical copies number and target gene B to be detected；Adjustment concentration makes the molar ratio of A and ABm in system close to 1:1 level builds the PCR reactions of two systems：The mutant fragments Am of reference gene A and A and the mutant fragments Bm for expanding target gene B and B are respectively used for amplifying, analyzes A in two systems:The molar ratio An and B of Am:The copy number N=X*Bn/An of the molar ratio Bn of Bm, then target gene B to be detected.The present invention is by way of adding in reference gene, and using simple regular-PCR technology, the quick copy number for judging target gene, method is ingenious, and as a result strong antijamming capability is stablized, of low cost, high resolution, is the breakthrough method of copy number of target genes identification.
Description
Technical field
The invention belongs to technical field of molecular biology, and in particular to judge the side of copy number of target genes in genome
Method.
Background technology
Genetically modified plants are to possess the plant from other species genes, which typically now refers to pass through
Recombinant DNA technology is manually inserted into other species genes to create the plant for possessing new features, such as has plant pest-resistant, anti-
The new features such as sick, degeneration-resistant, high yield, high-quality.
In the insertion Plant Genome that target gene can be random in genetically modified plants are produced, copy number and site are inserted into all
It is random, target gene more than 50% can be inserted into the form of singly copying in agriculture bacillus mediated plant transgene, and base
Because target gene is then based on polygenes copy insertion in the plant transgene of rifle mediation, but in the application in later stage and gene work(
In analyzing, it is necessary to be copied with single as experiment material, to ensure the reliable and stable of experimental result.So identification transgene copies
Number, which is one of transgenosis post analysis, to work.
At present, the authoritative method for analyzing copy number has southern blot, and experimental technique is with high requirements and high cost, radiation
Property isotope it is dangerous high, digoxin effect is undesirable；Other methods have a quantitative PCR, but quantifying PCR method and are not allowed
Really, because its maximum difference resolution ratio at least needs one times or more, the credibility of result cannot reach 100%.
The content of the invention
In order to solve the problems in the prior art, the purpose of the present invention is to provide target gene in a kind of judgement genome and copy
Several methods.
To achieve the above object, the technical solution adopted by the present invention is：
The method of copy number of target genes in genome is judged, with known copy number in the genome of species to be measured
Gene is denoted as the reference gene A that copy number is X as reference gene A；
A genetic fragment ABm is built, the mutant fragments Am of the reference gene A sequences containing identical copies number and is treated thereon
Detect the mutant fragments Bm of target gene B；
Add in genomic templates and ABm in PCR system, adjustment concentration makes the molar ratio of A and ABm in system close to 1:1
Level, build the PCR reactions of two systems：Pair of primers PA-F/PA-R is designed in first system for expanding internal reference base
Pair of primers PB-F/PB-R is designed because of the mutant fragments Am of A and A, in second system to be used to expand the prominent of target gene B and B
Become segment Bm, all by the use of the genome of identical mixing and the DNA of ABm segments as template, the expansion of PA-F/PA-R in two systems
Volume increase object is A/Am, and the amplified production of PB-F/PB-R is B/Bm, analyzes A in two systems:The molar ratio An and B of Am:Bm's
The copy number N=X*Bn/An of molar ratio Bn, then target gene B to be detected.
Further, the genetic fragment ABm is an a reference carrier ABm or DNA fragmentation ABm.
Preferably, four kinds of bases of the mutant fragments Am and mutant fragments Bm are mutated to be balanced, and mutational site number is more than 4
It is a, and the template after mutation and the template before mutation have identical PCR amplification efficiency.
Preferably, the molar ratio for making A and ABm in system is close to 1:1 method is：By the size for calculating genome
It is calculated with the molecular size where the concentration of genomic templates and the concentration of ABm and ABm.
Preferably, the computational methods of the An and Bn are obtained by high-flux sequence or generation sequencing approach.
Preferably, use generation sequencing approach obtain the method for An and Bn values for：By the amplification knot of two PCR systems respectively
Fruit carries out generation sequencing, obtained peak-data will be sequenced and submits to meter together as the homologous sequence of reference diversity sequence
Calculation machine program is compared, and counts the mean intensity in each sequence all differences site, the ratio of each sequence average signal strength
Represent the quantity ratio of the homologous sequence；The homologous sequence is A/Am sequences or B/Bm sequences.
Preferably, the primer PA-F/PA-R or primer PB-F/PB-R is with the corresponding homologous sequence
100% matches, and difference site scope is 4~30 in the amplification target DNA that amplification procedure is selected.
Preferably, the method that the computer program is compared includes：First calculated according to the reference diversity sequence of submission
All differences site, the specific base for recording these difference sites and position in the sequence；Then according to these location informations
The signal strength of 4 kinds of bases on these positions is found out in sequencing result file data, and is recorded；It counts on all differences site
The signal strength of different bases；The signal strength in each site is uniformed；Count each sequence all differences site
Mean intensity, the ratio of each sequence average signal strength represent the quantity ratio of each homologous sequence in starting system.
Compared with prior art, the beneficial effects of the invention are as follows：The present invention utilizes letter by way of adding in reference gene
Single regular-PCR technology, the quick copy number for judging target gene, method is ingenious, and as a result strong antijamming capability is stablized, cost
Cheap, high resolution is the breakthrough method that copy number of target genes is identified.
Description of the drawings
Fig. 1 is the pUC18-ABm carrier schematic diagrames built in embodiment.
Specific embodiment
Below in conjunction with the attached drawing in the present invention, technical scheme is clearly and completely described, it is clear that
Described embodiment is only part of the embodiment of the present invention, instead of all the embodiments.Based on the implementation in the present invention
Example, all other embodiment that those of ordinary skill in the art are obtained under the conditions of creative work is not made belong to
The scope of protection of the invention.
Embodiment 1
The present embodiment is to identify in transgenic paddy rice exemplified by the copy number of target gene：
Cytochrome C gene (Rice Cytochrome c Gene) is singly to copy in genome in known rice cell,
Reference gene A is one section of sequence on cytochrome C gene, SEQ ID NO.1 institutes in the sequence such as sequence table of reference gene A
Show, mutant fragments Am is as shown in SEQ ID NO.2 in sequence table；Target gene B is the common resistance base of transgenosis
Cause --- one section of sequence on hygromycin gene, as shown in SEQ ID NO.3 in sequence table, in mutant fragments Bm such as sequence table
Shown in SEQ ID NO.4；A genetic fragment ABm is built, as shown in Figure 1.
It is as follows：
The first step first builds pUC18-Am carriers
(1) synthesis is following a pair of for expanding the primer of Am, respectively as shown in sequence table SEQ ID NO.5~6：
Am(+):cagtAagcttcggaggattgtgtggttgat
Am(-):cgatGgatccctctctaaccatgtttaaga
The PCR reactions of 1 50ul system are done, composition is as follows：
Total:50ul
Sum:1
H2O:34ul
buffer:5ul
Mg2+:4ul
dNTP:2ul
Am+:2ul
Am-:2ul
taq:2U
rice genome:1ul
PCR programs are：
94℃ for 5min；
94 DEG C of for 30s, 50 DEG C of for 45s, 72 DEG C of for 25s, 30 cycle；
72 DEG C of for 10min, 16 DEG C of for 30min.
After amplification, respectively will：The electrophoresis segment of 420bp is cut, and is placed on progress colloidal sol recycling in same system, is returned
The kit specification that program is shown in specific producer is received, (recovery product is labeled as with the water dissolution recycling DNA of total volume 30ul：
RDNAA1), it is attached after detection is errorless with carrier.
(2) carrier digestion system：
total:20ul
Sum:1
H2O:12ul
Buffer:2ul
HindIII:1ul
BamHI:1ul
pUC18:4ul
Digestion program：37℃for 1h.
(3) rDNAA1 digestions system：
total:20ul
Sum:1
H2O:12 12ul
Buffer:2 2ul
HindIII:1 1ul
BamHI:1 1ul
rDNAA1:4ul
Digestion program：37℃for 1h.
The carrier digestion object of step (2) and this step recycling segment digestion products are merged one and reinstate PCR purification kits
Purifying (purified product is labeled as P-rDNAA1) is used for the coupled reaction of next step
(4) coupled reaction system
total:10ul
Sum:1
H2O:5.5ul
Buffer:1ul
T4-ligase:1ul
Coupled reaction program：20℃for 1h.
By connection product transformed competence colibacillus, step is：5-10ul connection products are converted into E. coli competent (see large intestine
Bacillus competence transformation standard method) conversion painting resistance plate, when 37 DEG C of cultures 12 are small, carry out bacterial plaque pcr identifications.By what is obtained
PUC18-Am carriers are further used for follow-up vector construction and finally obtain pUC18-ABm
Bm is cloned into pUC18-Am and obtains pUC18-ABm by second step
(1) synthesis is following a pair of for expanding the primer of Bm, respectively as shown in sequence table SEQ ID NO.7~8：
Bm(+):cagtGgatccagcgtctccgacctgatgca
Bm(-):cgatGaattcctcatcgagagcctgcgcga
The PCR reactions of 1 50ul system are done, composition is as follows：
Total:50ul
Sum:1
H2O:34ul
buffer:5ul
Mg2+:4ul
dNTP:2ul
Bm+:2ul
Bm-:2ul
taq:2U
hyg DNA:1ul
PCR programs are：
94℃ for 5min；
94 DEG C of for 30sec, 50 DEG C of for 45sec, 72 DEG C of for 29sec, 30 cycle；
72 DEG C of for 10min, 16 DEG C of for 30min.
After amplification, respectively will：The electrophoresis segment of 480bp is cut, and is placed on progress colloidal sol recycling in same system, is returned
The kit specification that program is shown in specific producer is received, (recovery product is labeled as with the water dissolution recycling DNA of total volume 30ul：
RDNAB1), it is attached after detection is errorless with carrier.
(2) carrier digestion system：
total:20ul
Sum:1
H2O:12ul
Buffer:2ul
BamHI:1ul
EcoRI:1ul
pUC18-Am:4ul
Digestion program：37℃for 1h.
(3) rDNAB1 digestions system
total:20ul
Sum:1
H2O:12ul
Buffer:2ul
BamHI:1ul
EcoRI:1ul
rDNAB1:4ul
Digestion program：37℃for 1h.
The carrier digestion object of step (2) and this step recycling segment digestion products are merged one and reinstate PCR purification kits
Purifying (purified product is labeled as P-rDNAB1) is used for the coupled reaction of next step
(4) coupled reaction system
total:10ul
Sum:1
H2O:5.5ul
Buffer:1ul
T4-ligase:1ul
Coupled reaction program：20℃for 1h.
By connection product transformed competence colibacillus, step is：5-10ul connection products are converted into E. coli competent (see large intestine
Bacillus competence transformation standard method) conversion painting resistance plate, when 37 DEG C of cultures 12 are small, carry out bacterial plaque pcr identifications.
3rd step, quantitative experiment
Rice genome 466000000bp, pUC18-ABm：3547bp.
1.31ug genomic DNAs and 0.01ng pUC18-ABm plasmids is taken to mix as use amplification masterplate.
Build following two pcr systems：
PCRA：
H2O:34ul
buffer:5ul
Mg2+:4ul
dNTP:2ul
Am(+):2ul
Am(-):2ul
taq:2U
Masterplate:1ul；
PCRB：
H2O:34ul
buffer:5ul
Mg2+:4ul
dNTP:2ul
Bm(+):2ul
Bm(-):2ul
taq:2U
Masterplate:1ul
35 cyclic amplifications, amplified production are respectively A/Am, B/Bm, analyze A in two systems:The molar ratio An of Am and
B:The copy number N=X*Bn/An of the molar ratio Bn of Bm, then target gene B to be detected.
The homologous sequence is A/Am sequences or B/Bm sequences.
The computational methods of the An and Bn are obtained by generation sequencing approach：By the amplification of two PCR systems respectively
Generation sequencing is carried out, sequencing respectively obtains two sequencing results file Cytochrome_c.ab1, hyg-hygm.ab1, sequencing knot
The peak-data figure that fruit is opened with data software is checked；Obtained peak-data and the institute as reference diversity sequence will be sequenced
It states homologous sequence SEQ ID NO.1~2 or SEQ ID NO.3~4 to submit to computer program together and be compared, comparison process
As shown in the following table 1~2：
Table 1
Note：Base _ A：The corresponding base in difference site in reference gene A (Cytochrome_c)；Base _ Am：Mutation
The corresponding base in difference site in segment Am (Cytochrome_c (m))；Peak value _ A：Reference gene A (Cytochrome_c)
Upper difference site base signal strength values；Peak value _ Am：Difference site base is believed on mutant fragments Am (Cytochrome_c (m))
Number intensity value；Homogenization value _ A：On reference gene A (Cytochrome_c) after the homogenization of difference site base signal strength
Value；Homogenization value _ Am：Value on mutant fragments Am (Cytochrome_c (m)) after the homogenization of difference site base signal strength.
Table 2
Note：Base _ B：The corresponding base in difference site in target gene B (hyg)；Base _ Bm：Mutant fragments Bm (hyg
(m)) the corresponding base in difference site in；Peak value _ B：Difference site base signal strength values on target gene B (hyg)；Peak
Value _ Bm：Difference site base signal strength values on mutant fragments Bm (hyg (m))；Homogenization value _ B：On target gene B (hyg)
Value after the base signal strength homogenization of difference site；Homogenization value _ Bm：Difference site base on mutant fragments Bm (hyg (m))
Value after signal strength homogenization.
All differences sites is calculated according to the reference diversity sequence of submission, record these difference sites specific base and
Position in sequence, A/Am sequences obtain totally 14 difference sites, B/Bm sequences obtain totally 21 difference sites (with strain E35-
Exemplified by 2)；Then the signal for finding out 4 kinds of bases on these positions in sequencing result file data according to these location informations is strong
Degree, and record；Count the signal strength of different bases on all differences site；The signal strength in each site is carried out homogeneous
Change；The mean intensity in each sequence all differences site is counted, the ratio of each sequence average signal strength represents starting system
In each homologous sequence quantity ratio.
Result of calculation shows A：Relative populations ratio=1.521 of Am；
B:Relative populations ratio=0.755 of Bm；
This result of calculation can automatically generate on the site：http://39.108.2.122/.
After obtaining An, Bn, then the copy number N=X*Bn/An of target gene B to be detected.
T0 is for transgenic rice plant analysis result：
| Rice strain | E35-2 | E35-3 | E35-4 | E35-5 | E35-6 | E35-7 | E35-8 |
| An | 1.521 | 1.02 | 0.97 | 0.957 | 0.99 | 1.02 | 1.1 |
| Bn | 0.755 | 0.985 | 0.49 | 1.445 | 0.49 | 0.485 | 1.05 |
| N | 0.49665 | 0.965686 | 0.505155 | 1.509927 | 0.494949 | 0.47549 | 0.954545 |
| As a result judge | Single copy | Double copies | Single copy | 3 copies | Single copy | Single copy | Double copies |
Note：For T0 for transfer-gen plant because homozygous without carrying out, also there is no homologue in same position
Be inserted into exogenous sequences simultaneously, and cytochrome C gene be in the cell single copy (refer on a set of chromosome only there are one
Copy), but rice is 2 times of bodies, so one is intracellular there are two cytochrome C gene segment, when target gene and cell
Explanation has transgenic homozygote there are one insertion point in genome in the offspring generated when the ratio of pigment C is 0.5,
So that two gene molecules containing target gene into the cell.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
Understanding without departing from the principles and spirit of the present invention can carry out these embodiments a variety of variations, modification, replace
And modification, the scope of the present invention is defined by the appended.
SEQUENCE LISTING
<110>Wuhan Ai Deshi bio tech ltd
<120>Judge the method for copy number of target genes in genome
<130>
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 420
<212> DNA
<213>Reference gene A on rice cytochrome C gene
<400> 1
cggaggattg tgtggttgat gatgagatcc atgtttgtac tctttcaggt ccaaacttga 60
atggtctgtt tggaaggcag tcaggtacca cccctggtta ttcctactct acggccaaca 120
agaacatggc tgtgatctgg gaggagaaca cactttatga ctacctgctt aatcctaaga 180
aggtatgatt aatgataacg ttggccacct gagttctata attttacttg tagcctgcta 240
ttgtttcctt ccttttagcg tgcacagggt cggaactaaa tgttggggtt ttccagcctg 300
ttgttatttt gtattcctgt tgcttgtgac actttagagt aatcttcatg ctatctagtt 360
ttctgtattg gccgtccttt accatacaaa acttcaaaac tcttaaacat ggttagagag 420
<210> 2
<211> 420
<212> DNA
<213>The mutant fragments Am of reference gene A
<400> 2
cggaggattg tgtggttgat gatgagatcc atgtttgtac tctttcaggt ccaaacttga 60
atggtctgtt tggaagccag tcaggtacca gccctggtta tacctactct tcggccaact 120
acaacatgcc tgtgatgtgg gaggagatca cactttatga gtacctgcta aatcctaagt 180
aggtatgttt aatgataacc ttggccacct gagttctata attttacttg tagcctgcta 240
ttgtttcctt ccttttagcg tgcacagggt cggaactaaa tgttggggtt ttccagcctg 300
ttgttatttt gtattcctgt tgcttgtgac actttagagt aatcttcatg ctatctagtt 360
ttctgtattg gccgtccttt accatacaaa acttcaaaac tcttaaacat ggttagagag 420
<210> 3
<211> 480
<212> DNA
<213>Target gene B on hygromycin gene
<400> 3
agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat 60
gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat 120
cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt 180
ggggagttta gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg 240
caagacctgc ctgaaaccga actgcccgct gttctacaac cggtcgcgga ggctatggat 300
gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga 360
atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat 420
cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag 480
<210> 4
<211> 480
<212> DNA
<213>The mutant fragments Bm of target gene B
<400> 4
agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat 60
gtaggagcgc gtggatatga cctgcgggtt aatagctccg ccgatggttt gtacatagat 120
cgatatgttt ttcggcagtt tgcatgggcc gcgcacccga ttgcggaact gcttgagatt 180
ggggtgttta gcgagagcct gacctattgc atcacccgcc gtgcactggg tgtcacgatg 240
caagacctgc ctgaaagcga actgcccgct gttcttcaac cggtcgcgga ggctatggat 300
gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga 360
atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat 420
cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag 480
<210> 5
<211> 30
<212> DNA
<213>Artificial sequence Am (+)
<400> 5
cagtaagctt cggaggattg tgtggttgat 30
<210> 6
<211> 30
<212> DNA
<213>Artificial sequence Am (-)
<400> 6
cgatggatcc ctctctaacc atgtttaaga 30
<210> 7
<211> 30
<212> DNA
<213>Artificial sequence Bm (+)
<400> 7
cagtggatcc agcgtctccg acctgatgca 30
<210> 8
<211> 30
<212> DNA
<213>Artificial sequence Bm (-)
<400> 8
cgatgaattc ctcatcgaga gcctgcgcga 30
Claims (7)
1. judge the method for copy number of target genes in genome, which is characterized in that with one in the genome of species to be measured
Know that the gene of copy number as reference gene A, is denoted as the reference gene A that copy number is X；
A genetic fragment ABm is built, thereon the mutant fragments Am of the reference gene A sequences containing identical copies number and to be detected
The mutant fragments Bm of target gene B；
Add in genomic templates and ABm in PCR system, adjustment concentration makes the molar ratio of A and ABm in system close to 1:1 water
It is flat, build the PCR reactions of two systems：Pair of primers PA-F/PA-R is designed in first system for expand reference gene A and
The mutant fragments Am of A designs the mutant fragments that pair of primers PB-F/PB-R is used to expand target gene B and B in second system
Bm, all by the use of the genome of identical mixing and the DNA of ABm segments as template, the amplified production of PA-F/PA-R in two systems
For A/Am, the amplified production of PB-F/PB-R is B/Bm, analyzes A in two systems:The molar ratio An and B of Am:The molar ratio of Bm
The copy number N=X*Bn/An of value Bn, then target gene B to be detected.
2. the method according to claim 1 for judging copy number of target genes in genome, which is characterized in that the mutation
Four kinds of bases of segment Am and mutant fragments Bm are mutated to be balanced, and mutational site number is more than 4, and the template after mutation and mutation
Preceding template has identical PCR amplification efficiency.
3. the method according to claim 1 for judging copy number of target genes in genome, which is characterized in that described to make body
The molar ratio of A and ABm is close to 1 in system:1 method is：By calculate the size of genome and the concentration of genomic templates and
Molecular size where the concentration and ABm of ABm is calculated.
4. it is according to claim 1 judge genome in copy number of target genes method, which is characterized in that the An and
The computational methods of Bn are obtained by high-flux sequence or generation sequencing approach.
5. the method according to claim 4 for judging copy number of target genes in genome, which is characterized in that using a generation
The method that sequencing approach obtains An and Bn values is：The amplification of two PCR systems respectively is subjected to generation sequencing, will be sequenced
To peak-data and homologous sequence as reference diversity sequence submit to computer program together and be compared, statistics is every
The mean intensity in a sequence all differences site, the ratio of each sequence average signal strength are the number for representing the homologous sequence
Measure ratio；The homologous sequence is A/Am sequences or B/Bm sequences.
6. the method according to claim 5 for judging copy number of target genes in genome, which is characterized in that the primer
PA-F/PA-R or primer PB-F/PB-R is 100% matching with the corresponding homologous sequence, the expansion that amplification procedure is selected
It is 4~30 to increase difference site scope in target DNA.
7. the method according to claim 5 or 6 for judging copy number of target genes in genome, which is characterized in that described
The method that computer program is compared includes：All differences site is first calculated according to the reference diversity sequence of submission, records this
The specific base in a little difference sites and position in the sequence；Then according to these location informations in sequencing result file data
The signal strength of 4 kinds of bases on these positions is found out, and is recorded；Count the signal strength of different bases on all differences site；
The signal strength in each site is uniformed；Count the mean intensity in each sequence all differences site, each sequence average
The ratio of signal strength represents the quantity ratio of each homologous sequence in starting system.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101012482A (en) * | 2007-02-12 | 2007-08-08 | 中国农业大学 | Method for sifting differentia site and flank sequence of genom DNA |
| CN103525922A (en) * | 2013-09-29 | 2014-01-22 | 上海辰山植物园 | Endogenous reference gene suitable for brachypodium distachyon exogenous gene detection and copy number analysis, and application thereof |
| CN103534356A (en) * | 2011-02-21 | 2014-01-22 | 凯杰有限公司 | Method for quantifying human dna using an internal control |
-
2017
- 2017-12-29 CN CN201711482313.5A patent/CN108103154A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101012482A (en) * | 2007-02-12 | 2007-08-08 | 中国农业大学 | Method for sifting differentia site and flank sequence of genom DNA |
| CN103534356A (en) * | 2011-02-21 | 2014-01-22 | 凯杰有限公司 | Method for quantifying human dna using an internal control |
| CN103525922A (en) * | 2013-09-29 | 2014-01-22 | 上海辰山植物园 | Endogenous reference gene suitable for brachypodium distachyon exogenous gene detection and copy number analysis, and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| JIAYU DING等: "Validation of a Rice Specific Gene, Sucrose Phosphate Synthase, Used as the Endogenous Reference Gene for Qualitative and Real-Time Quantitative PCR Detection of Transgenes", 《J.AGRIC.FOOD CHEM.》 * |
| 杨立桃等: "利用实时荧光定量PCR方法分析转基因水稻外源基因拷贝数", 《中国食品卫生杂志》 * |
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