CN100510107C - Method for preparing internal standard of molecular weight, and internal standard of molecular weight prepared by using the method - Google Patents
Method for preparing internal standard of molecular weight, and internal standard of molecular weight prepared by using the method Download PDFInfo
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- CN100510107C CN100510107C CNB2007100647134A CN200710064713A CN100510107C CN 100510107 C CN100510107 C CN 100510107C CN B2007100647134 A CNB2007100647134 A CN B2007100647134A CN 200710064713 A CN200710064713 A CN 200710064713A CN 100510107 C CN100510107 C CN 100510107C
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Abstract
This invention relates to a method for preparing molecular weight internal standard. The method comprises: immobilizing upstream primer 5'GCGAAACCCGACAGGACTA3' with pUC18 plasmid as the template, designing downstream primers with a certain distance, and proliferating to obtain multiple nucleotide segments. The method obtains a series of segments with different lengths by immobilizing the upstream primer and designing the downstream primers in sequence. The traditional method for synthesizing primers by fluorescent labeling has such disadvantages as high cost and short preservation time. The method in this invention synthesizes the upstream immobilized primer only, which can largely lower the primer synthesis cost and production cost. Since the upstream primer is the same, the lengths and annealing temperatures of the downstream primers are similar, and the proliferation conditions for all segments are identical, which can facilitate easy operation (all proliferations are performed on the same gene proliferation apparatus with the same reaction program), saved time, high working efficiency, and large-scale production.
Description
Technical field
The invention belongs to biological technical field, particularly relate to mark and preparation method thereof in a kind of molecular weight.
Background of invention
Since the Human Genome Project began, genescan and genotyping technique development were rapidly.In the Disease-causing gene examination, the chain and related somatotype of disease gene, all widespread uses of aspect such as disease early diagnosiss such as tumour, medical jurisprudence individual recognition.The important means of genotyping technique are utilized dna sequencing instrument or the segmental molecular weight of genetic analyzer analyzing gene exactly.Need a molecular weight standard to come sample is calculated during this quasi-instrument analyzing molecules amount, just mark in the molecular weight is called molecular weight internal reference (Internal Lane Standard) again.
Compare with traditional molecular weight standard (marker), mark belongs to internal reference in the molecular weight, and traditional molecular weight standard belongs to outer contrast, and internal reference and testing sample are at same swimming lane electrophoresis, and the two characteristics of motion is in full accord, so collation is more accurate.In addition, the target tolerance range is higher in the molecular weight, the electrophoretic medium of tradition molecular weight standard is sepharose and the lower polyacrylamide gel (PAGE) of resolving power often, resolving power is about tens bp, and the target electrophoretic medium is the PAGE or the capillary electrophoresis glue of order-checking in the molecular weight, and resolving power can reach 1 bp, therefore, this just requires interior target self molecular weight of molecular weight very accurate, and the difference of 1 base can not be arranged.In addition, owing to testing sample and molecular weight standard need be distinguished, mark need carry out fluorescent mark in the molecular weight, could be discerned by instrument, and the fluorescence molecule of mark needs different marks when analyzing the sample of isolabeling not, and traditional molecular weight standard does not have fluorescent mark, can't be used as internal reference.
Because fluorescently-labeled molecular weight requires molecular weight to be accurate to 1 Nucleotide, also needs to have fluorescent mark, its making method is also very big with traditional molecular weight standard difference.Conventional preparation molecular weight standard method is difficult to obtain in a large number, marks in the fluorescent tag molecule amount accurately.
Summary of the invention
The present invention is directed to the deficiency in the above-mentioned field, provide a kind of economy fast to prepare calibration method in the molecular weight, mark product in a kind of brand-new molecular weight obtained by this method also is provided simultaneously.
Target preparation method in a kind of molecular weight is a template with the pUC18 plasmid, and fixedly upstream primer 5 ' GCGAAACCCGACAGGACTA3 ' chooses respective distance design downstream primer, and amplification obtains a plurality of nucleotide fragments.
Described downstream primer is respectively
70R：5′ACAGGAGAGCGCACGAGG 3′：
80R：5′CAGGGTCGGAACAGGAGAG 3′；
100R：5′GACAGGTATCCGGTAAGCGG 3′；
120R：5′TTCCCGAAGGGAGAAAGGC 3′；
140R：5′GCTATGAGAAAGCGCCACG 3′；
160R：5′CTGAGATACCTACAGCGTGAGC 3′；
180R：5′AGCGAACGACCTACACCGA 3′；
200R：5′GTGCACACAGCCCAGCTTG 3′；
240R：5′TACCGGATAAGGCGCAGC 3′；
280R：5′GATAAGTCGTGTCTTACCGGGT 3′；
320R：5′CGCTCTGCTAATCCTGTTACCA 3′；
360R：5′GCCACCACTTCAAGAACTCTGT 3′；
400R：5′CAGATACCAAATACTGTTCTTCTAGT 3′；
450R：5′ATCAAGAGCTACCAACTCTTTTTCC 3′；
490R：5′ACAAAAAAACCACCGCTACCA 3′；
500R：5′CTGCTTGCAAACAAAAAAACC 3′；
Amplification obtains 70bp, 80bp, 100bp, 120bp, 140bp, 160bp, 180bp, 200bp, 240bp, 280bp, 320bp, 360bp, 400bp, 450bp, 490bp, 500bp fragment respectively.
Described amplification reaction condition is: 94 ℃ of 2min; 30cycles:94 ℃ of 20sec, 58 ℃ of 40sec, 72 ℃ of 40sec; 70 ℃ of 10min.
Described upstream primer has fluorescent marker.
Described fluorescent marker is ROX, FAM, HEX, JOE, TMR, TET, VIC, LIZ or Cy5.
Mark in the molecular weight that method for preparing obtains.
The present invention passes through a widely used plasmid vector (pUC18) as dna profiling.With P rimer Premier 5.0 software analysis pUC18 plasmid complete sequences, (seeing appendix SEQ ID NO1) found at its 927bp place to the 1425bp place, the ATGC uniform content, there is not special secondary structure, choose the zone about this 500bp, immobilized primer 5 ' GCGAAACCCGACAGGACTA3 ' of (5 ') design designs a series of primers in the downstream in the upstream, and making amplified production is a series of nucleotide fragments.The present invention passes through fixedly upstream primer, and the method that designs downstream primer successively downwards obtains a series of fragments that are uneven in length.Can reduce cost like this, save time, increase work efficiency.
Upstream primer 5 ' GCGAAACCCGACAGGACTA3 ', 58 ℃ of annealing temperatures.With the upstream primer is starting point, look for the terminal point of downstream primer in its downstream respective distance, the length of downstream primer and sequence to guarantee preferably specificity simultaneously annealing temperature to catch up with the trip primer approaching, GC content is between 20-40%, do not have complicated second level outcome, can not form dimer with upstream primer.The present invention selects that the length of nucleotide fragments is respectively 70,80,100,120,140,160,180,200,240,280,320,360,400,450,490,500bp, determines that finally the downstream primer sequence is as follows:
Primer title primer sequence (5 '-3 ')
70R 5′ACAGGAGAGCGCACGAGG 3′
80R 5′CAGGGTCGGAACAGGAGAG 3′
100R 5′GACAGGTATCCGGTAAGCGG 3′
120R 5′TTCCCGAAGGGAGAAAGGC 3′
140R 5′GCTATGAGAAAGCGCCACG 3′
160R 5′CTGAGATACCTACAGCGTGAGC 3′
180R 5′AGCGAACGACCTACACCGA 3′
200R 5′GTGCACACAGCCCAGCTTG 3′
240R 5′TACCGGATAAGGCGCAGC 3′
280R 5′GATAAGTCGTGTCTTACCGGGT 3′
320R 5′CGCTCTGCTAATCCTGTTACCA 3′
360R 5′GCCACCACTTCAAGAACTCTGT 3′
400R 5′CAGATACCAAATACTGTTCTTCTAGT 3′
450R 5′ATCAAGAGCTACCAACTCTTTTTCC 3′
490R 5′ACAAAAAAACCACCGCTACCA 3′
500R 5′CTGCTTGCAAACAAAAAAACC 3′
By groping, determine that finally amplification reaction condition is: 94 ℃ of 2min; 30cycles:94 ℃ of 20sec, 58 ℃ of 40sec, 72 ℃ of 40sec; 70 ℃ of 10min.Each fragment can both have the specificity that higher amplification efficiency is become reconciled under this condition.
Because upstream primer unanimity, the length of downstream primer and annealing temperature and upstream are close, so whole fragment amplification conditions are basically identical all, can on same gene-amplificative instrament, increase simultaneously with same response procedures, feasible operation is easier, save time, improved working efficiency, be suitable for mass preparation.
The fluorescent marker that the present invention prepares target process middle and upper reaches primer in the molecular weight can be ROX, also can the time be FAM (Fluoresceincarboxylic acid), HEX (chlordene-6-methyl fluorescein), JOE (4,5 two chloro-6-Fluoresceincarboxylic acid), TMR (4-methyl-6-carboxyl-rhodamine) or TET, VIC, LIZ, fluorescent markers such as Cy5, and prepare mark in the molecular weight with this method.Because the synthetic cost of fluorescent dye primer is higher, the shelf time is limited, so the inboardend primer of synthetic upstream only can reduce synthetic cost of primer and production cost so greatly.
The present invention prepares a kind of mark in the fluorescently-labeled nucleic acid molecular weight that has, by length be 70,80,100,120,140,160,180,200,240,280,320,360,400,450,490,16 double chain DNA fragments of 500bp form, each fragment has a DNA chain to have fluorescent marker, through after the electrophoretic separation, laser excitation produces exciting light and can be detected by fluorescence detector (as the FMBIO of Hitachi fluorescence detector, ABI 3100 genetic analyzers etc.), is used for analyzing the size of target DNA fragment in the same swimming lane.It is the molecular weight of 70bp to the nucleic acid fragment between the 500bp that the interior mark of this molecular weight can be used for calculating and proofreading length.Because it is fluorescein-labelled that product has, need pH greater than 8.0 buffer system in cryogenic freezing preserve, and can not multigelation.Low pH environment and multigelation can cause fluorescent marker to come off, thereby cause that detection signal reduces the sensitivity variation.
Marking in the molecular weight that utilizes this method to make is double chain DNA molecule, wherein has only one to have fluorescent marker, needs sex change to become single chain molecule before carrying out denaturing gel electrophoresis.General method is to add deionized formamide, and 95 ℃ of heating sex change in 3-5 minute placed cooled on ice rapidly 3 minutes.
The interior mark of the present invention's preparation can be used for denaturing polyacrylamide gel electrophoresis and capillary electrophoresis.
Description of drawings
Fig. 1: have mark electrophoretogram in the fluorescently-labeled molecular weight of ROX
Fig. 2: detect the somatotype result after adopting the present invention as mark and Goldeneye 16A allelic ladder co-electrophoresis in the molecular weight
Fig. 3: the ILS600 that adopts Promega company detects the somatotype result as behind mark in the molecular weight and the Goldeneye 16A allelic ladder co-electrophoresis
Embodiment
Mark in the embodiment 1 preparation molecular weight
1.1 the upstream primer of design upstream primer and synthetic ROX mark.
The dna profiling of selecting for use is pUC18 plasmid (Takara company, article No. D3218).Selecting the 927bp 5 ' GCGAAACCCGACAGGACTA3 ' of place is upstream primer, 58 ℃ of annealing temperatures.ABI394 nucleic acid synthesizer synthetic primer, and at 5 ' of primer ROX mark, HPLC method purifying primer.
1.2 design downstream primer according to fragment length.
Select a series of fragment 70,80,100,120,140,160,180,200,240,280,320,360,400,450,490,500 totally 16 fragments that are uneven in length.Can also be a plurality of nucleotide fragments that are uneven in length between 927-1425bp.ABI394 nucleic acid synthesizer synthetic primer, the PAGE purifying.Downstream primer selects sequence as follows:
Primer title primer sequence (5 '-3 ')
70R 5′ACAGGAGAGCGCACGAGG 3′
80R 5′CAGGGTCGGAACAGGAGAG 3′
100R 5′GACAGGTATCCGGTAAGCGG 3′
120R 5′TTCCCGAAGGGAGAAAGGC 3′
140R 5′GCTATGAGAAAGCGCCACG 3′
160R 5′CTGAGATACCTACAGCGTGAGC 3′
180R 5′AGCGAACGACCTACACCGA 3′
200R 5′GTGCACACAGCCCAGCTTG 3′
240R 5′TACCGGATAAGGCGCAGC 3′
280R 5′GATAAGTCGTGTCTTACCGGGT 3′
320R 5′CGCTCTGCTAATCCTGTTACCA 3′
360R 5′GCCACCACTTCAAGAACTCTGT 3′
400R 5′CAGATACCAAATACTGTTCTTCTAGT 3′
450R 5′ATCAAGAGCTACCAACTCTTTTTCC 3′
490R 5′ACAAAAAAACCACCGCTACCA 3′
500R 5′CTGCTTGCAAACAAAAAAACC 3′
1.3 grope amplification reaction condition, obtain product and detection with this condition amplification, amplified reaction carries out on Bio-Rad iCycler gene-amplificative instrament.
Synthetic respectively above-mentioned upstream primer and downstream primer (ABI 394 nucleic acid synthesizers are finished) are diluted to 10 μ M with primer with TE, grope primer concentration in the reaction system, template DNA concentration and Taq enzyme dosage, finally determine reaction system compound method such as following table:
| Reacted constituent | Add volume (μ l) |
| ddH 2O | 6 |
| 10 * reaction buffer | 1 |
| dNTP(2.5mM) | 0.8 |
| Upstream primer (10 μ M) | 0.25 |
| Downstream primer (2.5 μ M) | 1 |
| pUC18(1ng/μl) | 1 |
| Warm start Taq enzyme (5U/ μ l) | 0.2 |
| Cumulative volume | 10 |
Annealing temperature is by being provided with thermograde, and from 54,56,58,60 ℃ of amplifications respectively, detection reaction efficient and atopic determine that finally reaction conditions is: 94 ℃ of 2min; 30cycles:94 ℃ of 20sec, 58 ℃ of 40sec, 72 ℃ of 40sec; 70 ℃ of 10min.Each fragment can both have the specificity that higher amplification efficiency is become reconciled under this condition.
1.4 adjust the product amount, make each band peak equalization, the results are shown in Figure 1.
Make it molar ratio such as reach by the content of adjusting each band, peakedness ratio is than homogeneous, and ABI 3100 genetic analyzers detect.Detection method is as follows:
1) get 0.5 μ l and mix with 9.5 μ l Hi-Di methane amides, 95 ℃ of sex change 3 minutes place on ice immediately;
2) the ABI3100 genetic analyzer detects (parameter setting: 1 kilovolt of sample introduction voltage, sample injection time 15 seconds, 15 kilovolts of electrophoretic voltages, electrophoresis time 1400 seconds)
3) each fragment peak height is greater than 500 RFU, and difference in height is less than 10% between each fragment.
1.5 product purification and freezing preservation.Contain albumen and salt ion in the above-mentioned process detection peak isostatic dna fragmentation mixture, can cause the dna fragmentation degraded, be unfavorable for prolonged preservation, therefore need be purified removal impurity.Utilize day centrifugal column type DNA purification kit of root biochemical corp to finish, working method is as follows:
1) each reaction product is mixed together; Estimate the volume of product, to wherein add 10 times of volumes in conjunction with liquid, gentle abundant mixing.
2) previous step gained solution is added in the adsorption column (adsorption column is put into collection tube), room temperature was placed 2 minutes, the centrifugal 30-60 of 13000rpm second outwelled the waste liquid in the collection tube, and adsorption column is put into collection tube.
3) in adsorption column, add the rinsing liquid of 700 μ l, the centrifugal 30-60 of 13000rpm second outwell the waste liquid in the collection tube, adsorption column is put into collection tube.
4) in adsorption column, add the rinsing liquid of 500 μ l, the centrifugal 30-60 of 13000rpm second, outwell waste liquid.
5) adsorption column is relay in the recovery collector, centrifugal 2 minutes of 13000rpm removes rinsing liquid as far as possible.Adsorption column is placed room temperature and 58 ℃ of incubator numbers minute, thoroughly dry, influence next step experiment to prevent residual rinsing liquid.
6) take out adsorption column, put into a clean centrifuge tube, to the elution buffer of an amount of 65-70 ℃ of water-bath preheating of the unsettled dropping in adsorption film mid-way, room temperature was placed 2 minutes.Centrifugal 1 minute of 13000rpm collects solution.Promptly contain in the solution as 16 dna fragmentations of target in the molecular weight.
Mark the analyzing gene fragment in the molecular weight of embodiment 2. usefulness ROX marks and check accuracy
Utilize it to carry out molecular weight calibration respectively by FAM, HEX, the allelic ladder (Goldeneye16A identity authentication reagent constituents of basic point cognitive techniques (Beijing) company limited one of) of the length of TMR mark between 100-480bp, comprise 210 allelotrope fragments altogether, calculate each segmental mean size and variance.Control experiment adopts the product I LS 600 (article No. DG2611) of U.S. Promega company to analyze this allelic ladder.ILS 600 is made up of the double-stranded DNA of 22 60-600bp of ROX mark, and wherein size is the 20bp of being separated by between the fragment of 60-200bp, the 25bp of being separated by between the fragment of 200-500bp, the 50bp of being separated by between the fragment of 500-600bp.
Electrophoresis detection is finished on ABI 3100 genetic analyzers, electrophoretic voltage 15kV, collection time 1400 seconds.Sample is while electrophoresis in 12 swimming lanes respectively, finishes back segmental molecular weight of each allelotrope in each swimming lane of gene mapper 3.2 software analysis, and it is poor to calculate each segmental molecular weight standard then.
The examination and test of products standard that provides according to Promega company as can be known, analyze the segment size of allelotrope ladder with interior mark, repeat sample 12 times, analyze each genotypic 12 numerical value in each site among the ladder, take the mean and base of calculation deviation (SD), the SD value should be less than 0.1 base, but Penta D, Penta E, the SD value in FGA and D18S51 site can be between 0.1 to 0.2 base.In the internal standard line equation that is calculated with GeneScan software and Local Southern Method method, R
2Value should be greater than 0.9999.
Analytical results shows, analyzes 12 results of allelic ladder with the present invention, and the standard deviation of each fragment length is all less than 0.10 (between 0.03-0.07), R
2Value be 0.999999 (No. 3 swimming lanes are analyzed collection of illustrative plates and seen Fig. 2).The result who analyzes with ILS 600 similarly, the standard deviation of each sample is less than 0.10 (between 0.03-0.07), R
2Value be 0.999999 (No. 3 swimming lanes are analyzed collection of illustrative plates and seen Fig. 3).As seen, the prepared interior mark of the present invention has reached the examination and test of products index of Promega company, can be used for the molecular weight check and correction.
Appendix:
SEQ ID NO 1 (complete sequence of pUC18 plasmid)
1 TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG
51 GAGACGGTCA CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG
101 TCAGGGCGCG TCAGCGGGTG TTGGCGGGTG TCGGGGCTGG CTTAACTATG
151 CGGCATCAGA GCAGATTGTA CTGAGAGTGC ACCATATGCG GTGTGAAATA
201 CCGCACAGAT GCGTAAGGAG AAAATACCGC ATCAGGCGCC ATTCGCCATT
251 CAGGCTGCGC AACTGTTGGG AAGGGCGATC GGTGCGGGCC TCTTCGCTAT
301 TACGCCAGCT GGCGAAAGGG GGATGTGCTG CAAGGCGATT AAGTTGGGTA
351 ACGCCAGGGT TTTCCCAGTC ACGACGTTGT AAAACGACGG CCAGTGCCAA
401 GCTTGCATGC CTGCAGGTCG ACTCTAGAGG ATCCCCGGGT ACCGAGCTCG
451 AATTCGTAAT CATGGTCATA GCTGTTTCCT GTGTGAAATT GTTATCCGCT
501 CACAATTCCA CACAACATAC GAGCCGGAAG CATAAAGTGT AAAGCCTGGG
551 GTGCCTAATG AGTGAGCTAA CTCACATTAA TTGCGTTGCG CTCACTGCCC
601 GCTTTCCAGT CGGGAAACCT GTCGTGCCAG CTGCATTAAT GAATCGGCCA
651 ACGCGCGGGG AGAGGCGGTT TGCGTATTGG GCGCTCTTCC GCTTCCTCGC
701 TCACTGACTC GCTGCGCTCG GTCGTTCGGC TGCGGCGAGC GGTATCAGCT
751 CACTCAAAGG CGGTAATACG GTTATCCACA GAATCAGGGG ATAACGCAGG
801 AAAGAACATG TGAGCAAAAG GCCAGCAAAA GGCCAGGAAC CGTAAAAAGG
851 CCGCGTTGCT GGCGTTTTTC CATAGGCTCC GCCCCCCTGA CGAGCATCAC
901 AAAAATCGAC GCTCAAGTCA GAGGTGGCGA AACCCGACAG GACTATAAAG
951 ATACCAGGCG TTTCCCCCTG GAAGCTCCCT CGTGCGCTCT CCTGTTCCGA
1001 CCCTGCCGCT TACCGGATAC CTGTCCGCCT TTCTCCCTTC GGGAAGCGTG
1051 GCGCTTTCTC ATAGCTCACG CTGTAGGTAT CTCAGTTCGG TGTAGGTCGT
1101 TCGCTCCAAG CTGGGCTGTG TGCACGAACC CCCCGTTCAG CCCGACCGCT
1151 GCGCCTTATC CGGTAACTAT CGTCTTGAGT CCAACCCGGT AAGACACGAC
1201 TTATCGCCAC TGGCAGCAGC CACTGGTAAC AGGATTAGCA GAGCGAGGTA
1251 TGTAGGCGGT GCTACAGAGT TCTTGAAGTG GTGGCCTAAC TACGGCTACA
1301 CTAGAAGAAC AGTATTTGGT ATCTGCGCTC TGCTGAAGCC AGTTACCTTC
1351 GGAAAAAGAG TTGGTAGCTC TTGATCCGGC AAACAAACCA CCGCTGGTAG
1401 CGGTGGTTTT TTTGTTTGCA AGCAGCAGAT TACGCGCAGA AAAAAAGGAT
1451 CTCAAGAAGA TCCTTTGATC TTTTCTACGG GGTCTGACGC TCAGTGGAAC
1501 GAAAACTCAC GTTAAGGGAT TTTGGTCATG AGATTATCAA AAAGGATCTT
1551 CACCTAGATC CTTTTAAATT AAAAATGAAG TTTTAAATCA ATCTAAAGTA
1601 TATATGAGTA AACTTGGTCT GACAGTTACC AATGCTTAAT CAGTGAGGCA
1651 CCTATCTCAG CGATCTGTCT ATTTCGTTCA TCCATAGTTG CCTGACTCCC
1701 CGTCGTGTAG ATAACTACGA TACGGGAGGG CTTACCATCT GGCCCCAGTG
1751 CTGCAATGAT ACCGCGAGAC CCACGCTCAC CGGCTCCAGA TTTATCAGCA
1801 ATAAACCAGC CAGCCGGAAG GGCCGAGCGC AGAAGTGGTC CTGCAACTTT
1851 ATCCGCCTCC ATCCAGTCTA TTAATTGTTG CCGGGAAGCT AGAGTAAGTA
1901 GTTCGCCAGT TAATAGTTTG CGCAACGTTG TTGCCATTGC TACAGGCATC
1951 GTGGTGTCAC GCTCGTCGTT TGGTATGGCT TCATTCAGCT CCGGTTCCCA
2001 ACGATCAAGG CGAGTTACAT GATCCCCCAT GTTGTGCAAA AAAGCGGTTA
2051 GCTCCTTCGG TCCTCCGATC GTTGTCAGAA GTAAGTTGGC CGCAGTGTTA
2101 TCACTCATGG TTATGGCAGC ACTGCATAAT TCTCTTACTG TCATGCCATC
2151 CGTAAGATGC TTTTCTGTGA CTGGTGAGTA CTCAACCAAG TCATTCTGAG
2201 AATAGTGTAT GCGGCGACCG AGTTGCTCTT GCCCGGCGTC AATACGGGAT
2251 AATACCGCGC CACATAGCAG AACTTTAAAA GTGCTCATCA TTGGAAAACG
2301 TTCTTCGGGG CGAAAACTCT CAAGGATCTT ACCGCTGTTG AGATCCAGTT
2351 CGATGTAACC CACTCGTGCA CCCAACTGAT CTTCAGCATC TTTTACTTTC
2401 ACCAGCGTTT CTGGGTGAGC AAAAACAGGA AGGCAAAATG CCGCAAAAAA
2451 GGGAATAAGG GCGACACGGA AATGTTGAAT ACTCATACTC TTCCTTTTTC
2501 AATATTATTG AAGCATTTAT CAGGGTTATT GTCTCATGAG CGGATACATA
2551 TTTGAATGTA TTTAGAAAAA TAAACAAATA GGGGTTCCGC GCACATTTCC
2601 CCGAAAAGTG CCACCTGACG TCTAAGAAAC CATTATTATC ATGACATTAA
2651 CCTATAAAAA TAGGCGTATC ACGAGGCCCT TTCGTC
Claims (6)
1. the interior target preparation method of molecular weight is a template with the pUC18 plasmid, and fixedly upstream primer 5 ' GCGAAACCCGACAGGACTA3 ' chooses respective distance design downstream primer, and amplification obtains a plurality of nucleotide fragments.
2. target preparation method in the molecular weight according to claim 1, described downstream primer is respectively
70R：5′ACAGGAGAGCGCACGAGG 3′；
80R：5′CAGGGTCGGAACAGGAGAG 3′；
100R：5′GACAGGTATCCGGTAAGCGG 3′；
120R：5′TTCCCGAAGGGAGAAAGGC 3′；
140R：5′GCTATGAGAAAGCGCCACG 3′；
160R：5′CTGAGATACCTACAGCGTGAGC 3′；
180R：5′AGCGAACGACCTACACCGA 3′；
200R：5′GTGCACACAGCCCAGCTTG 3′；
240R：5′TACCGGATAAGGCGCAGC 3′；
280R：5′GATAAGTCGTGTCTTACCGGGT 3′；
320R：5′CGCTCTGCTAATCCTGTTACCA 3′；
360R：5′GCCACCACTTCAAGAACTCTGT 3′；
400R：5′CAGATACCAAATACTGTTCTTCTAGT 3′；
450R：5′ATCAAGAGCTACCAACTCTTTTTCC 3′；
490R：5′ACAAAAAAACCACCGCTACCA 3′；
500R：5′CTGCTTGCAAACAAAAAAACC 3′；
Amplification obtains 70bp, 80bp, 100bp, 120bp, 140bp, 160bp, 180bp, 200bp, 240bp, 280bp, 320bp, 360bp, 400bp, 450bp, 490bp, 500bp fragment respectively.
3. target preparation method in the molecular weight according to claim 2, the reaction conditions of described amplification is: 94 ℃ of 2min; 30cycles:94 ℃ of 20sec, 58 ℃ of 40sec, 72 ℃ of 40sec; 70 ℃ of 10min.
4. target preparation method in the molecular weight according to claim 1, described upstream primer has fluorescent marker.
5. target preparation method in the molecular weight according to claim 4, described fluorescent marker is ROX, FAM, HEX, JOE, TMR, LIZ or Cy5.
6. mark in the molecular weight that the target preparation method prepares in the arbitrary described molecular weight of claim 1 to 5.
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| CN106282180A (en) * | 2016-09-21 | 2017-01-04 | 无锡中德美联生物技术有限公司 | A kind of molecular weight internal standard and its preparation method and application |
| CN106566827B (en) * | 2016-11-07 | 2019-12-17 | 江苏苏博生物医学股份有限公司 | dye marking method |
| CN106868001A (en) * | 2017-04-10 | 2017-06-20 | 公安部第研究所 | One kind prepares calibration method in fluorescence molecule amount using rite-directed mutagenesis and enzyme incision technology |
| CN107964562B (en) * | 2017-12-14 | 2021-05-11 | 深圳华大法医科技有限公司 | Nucleic acid typing standard substance with fluorescent label and preparation method and application thereof |
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