CN106929589A - A kind of burnt sequencing analysis method based on coding multiple PCR products - Google Patents
A kind of burnt sequencing analysis method based on coding multiple PCR products Download PDFInfo
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Abstract
The invention discloses a kind of burnt sequencing analysis method based on coding multiple PCR products, comprise the following steps：(1) genomic DNA of sample is extracted；(2) it is template by encoding the multiplexed PCR amplification of primer with the genomic DNA of sample, obtains the PCR primer containing purpose fragment to be analyzed；(3) step (2) acquisition PCR primer is prepared into single stranded PCR products, the sequence information of coding region in the coding primer of burnt sequencing analysis single stranded PCR products；(4) sequence results are surveyed according to Jiao, it is determined that encode corresponding PCR primer whether there is, realizes the detection of the PCR primer characteristic DNA sequence.The burnt sequencing reaction that the method passes through one-time continuous, realize that multiplex PCR is possible to the qualitative and quantitative analysis of product, mix products to multiplex PCR are analyzed simultaneously, there is no relative length to limit, workload and the time of detection are reduced, is adapted to microbial rapid detection and the examination of the industries such as food, health.
Description
Technical field
The invention belongs to biological technical field, it is related to a kind of qualitative and quantitative analysis method of multiple PCR products, specifically relates to
And a kind of primer for encoding multiplex PCR, the then PCR primer of coding primer amplification and corresponding eventually through Jiao's sequencing decoding
The analytical technology that PCR primer whether there is.
Background technology
With progress, the improvement of people's living standards of society, people are food-safe, health care consciousness and requirement
More and more higher.How in food safety detection, cause the invasive organism of human diseases and food poisoning (such as：Salmonella
Bacterium, staphylococcus, streptococcus, vibrio parahemolyticus, proteus, Shigella, avian influenza virus, Aspergillus flavus and virus,
Foot and mouth disease virus etc.) it is varied, these food safety detections may relate to tens kinds of even a greater variety of microbial identifications,
But generally a kind of food is only possible to exist several or is not contaminated, how quickly the safety to food to be carried out
Accurately detection is to avoiding malignant event from occurring, and to the follow-up emergency processing of malignant event, fewer negative effect to society
Undoubtedly have great importance.And for example in medical diagnosis on disease and treatment, to the infectivity disease such as virus hepatitis, HPV
The diagnosis of disease, its Genotyping is likely to be related to types up to a hundred, but then may be only a kind of specific to specific certain patient
Genotype.Diagnosis that is how quick, accurate, realizing these communicable diseases cheaply, is the preciousness that treatment is won to patient
Time, the correct therapeutic modality of implementation, the medical expense of reduction patient have important practical significance.Above-mentioned detection is all referred to
The detection of nucleic acid molecules, i.e., find out the characteristic DNA template of necessary being in sample from multiple possible candidate feature DNA sequence dnas
Sequence.
Multiple PCR technique provides a kind of quick feasible way for the amplification of different DNA profilings, and multiplex PCR can be same
Different DNA profilings are obtained in reaction system, as long as can these different DNA profilings be implemented with analysis one by one, it becomes possible to realize many
The detection of component.However, the method based on gel electrophoresis analysis multiple PCR products depends on PCR primer, it is only different
(general two PCR fragment relative size needs to differ tens bases) just may be used in the case that PCR primer length is distinguished clearly
Can make correct judgement, therefore the resolving power of gel electrophoresis not high cause this cheap analysis method and can not expire
Sufficient quick detection is in the demand of diagnosis.Therefore, development one kind can accurately analyze these multiple different PCR primers, and there is reality to anticipate
Justice.
A kind of method for analyzing SNP site in multiple PCR primers is developed using biotech firm in the U.S.：
Multiple analysis kit.Using different length extension primer with comprising multiple SNP sites obtain PCR primer hybridize after, extend four kinds
The ddNTP of fluorescence labeling, and after similar to Sanger sequencing analysis, according to the length-specific designed for each site
Extension primer peak color can realize that each SNP site obtains parting.In this analysis method, the extension primer of length-specific can
To be considered as coding, extension primer one SNP site of correspondence of length-specific.This method can simultaneously analyze multiplex PCR
Product, with quick, accurate, relative low price feature.However, Sanger capillary sequencers are by the U.S.
Life departments under ThermoFisher house flags have monopoly power, expensive, and its analysis cost remains high, it is not easy to
Promote；And Sanger sequencings can only provide qualitative analysis, it is maximum it is not enough be because four kinds of dye baseline noises must influence, for
The qualitative analysis of PCR primer of the content less than 10% is also helpless.
Pyrosequencing (Pyrosequencing, also known as pyrosequencing techniques) is surveyed after the generation after Sanger sequencings
Sequence technology.Relative to Sanger sequencings, in addition to sequencing length is in weak tendency, its sequencing degree of accuracy is therewith quite but automatic
Change degree, operates advantageous and test limit in simple, price lower and with quantitative function, easily promotes, is very suitable for facing
Used in bed detection.However, Pyrosequencing does not have the mark different length that analysis part closing is capable of in Sanger sequencings
Sequencing primer extend fragment, similar toIt is mix products that coding techniques is not appropriate for analysis multiple PCR products,
Because the information of single PCR primer can cannot be obtained because of the interference of different PCR primer sequencing informations in Jiao's sequencing decoding.So
And, if partially enclosed, but the completely enclosed mode used unlike Sanger sequencings, then whenever encoding base is sequenced
After decoding, its extension primer is completely enclosed, does not continue to participate in the sequencing reaction that any nucleotides is added, i.e., to follow-up solution
Code sequencing reaction is without influence.But according to general full coding mode, at most also just can only the base different to four kinds carry out once
Coding, four different codings of acquisition.This undoubtedly limits encoded number so that the object of analysis is also limited by most 4.Such as
It not is full coding that really four different bases are used, but number of base is encoded, although be less than 4 in same position encoded number
It is individual, but can be encoded using multiple positions, can thus lift the number and the base for encoding primer sequence of coding
Number is more, and the number of coding is also more.
The content of the invention
Goal of the invention：For the problem that prior art is present, the present invention provides a kind of Jiao based on coding multiple PCR products
Sequencing analysis method.The method is the qualitative and quantitative analysis method to multiple PCR products, it is particularly possible to multiple in PCR primer
The simultaneous situation of PCR primer is detected.
Technical scheme：To achieve these goals, a kind of burnt sequencing based on coding multiple PCR products as described herein
Analysis method, comprises the following steps：
(1) blood, the genomic DNA of saliva equal samples are extracted；
(2) it is template by encoding the multiplexed PCR amplification of primer with the genomic DNA of sample, obtains containing to be analyzed
The PCR primer of purpose fragment；
(3) step (2) acquisition PCR primer is prepared into single stranded PCR products, the coding of burnt sequencing analysis single stranded PCR products
The sequence information of coding region in primer；
(4) sequence results are surveyed according to Jiao, it is determined that encode corresponding PCR primer whether there is, realizes the PCR primer feature
The detection of DNA sequence dna.
Wherein, step (2) the coding primer refers to one in one the two of purpose fragment to be analyzed PCR primer of amplification
It is coded of primer, and coding one PCR primer of correspondence；Another PCR primer is uncoded primer.
The coding primer is made up of three subregion sequences, i.e. specific hybridization regional sequence, Coding region sequence and logical
Use regional sequence；The specific hybridization regional sequence is used as specific PCR primer；The Coding region sequence is used to mark to determine PCR
Product；The general areas sequence is used for the sequencing primer of burnt sequencing analysis (burnt sequencing analysis are decoding) Coding region sequence
Hybridising region；The Coding region sequence is made up of 1 encoding base and several path bases, wherein positioned at coding region sequence
Last base of 3 ' ends is used to encode in row, referred to as encoding base, and sequence remaining base is referred to as path base.
The specific hybridization regional sequence of the coding primer is purpose fragment to be analyzed, be characterize object to be analyzed (such as certain
Kind of microorganism) one section of characteristic sequence, with uniqueness；Purpose fragment can be retrieved from existing database or document
Arrive.If containing object to be analyzed in sample, purpose fragment is contained in PCR primer；Otherwise, without purposeful in PCR primer
Fragment.The purpose fragment of one coding primer represents an object to be analyzed, and coding primer is at least 2, i.e., at least to be analyzed
Object be 2, constitute multiplex PCR.
Further, the coding primer is 10~20.
Wherein, another primer is not encoded in described two PCR primers, and 5 ' ends of the uncoded primer are repaiied by biotin
Decorations；The uncoded primer can constitute one the two of purpose fragment to be analyzed PCR primer of amplification with a coding primer,
Can be as another public primer of multiple coding primer.If having specific pcr template sequence in sample genome to be expanded
Increasing, then uncoded primer PCR product include one section of sequence with coding primer sequence complete complementary, and PCR primer length does not surpass
Cross 500bp.
Wherein, step (3) concretely comprise the following steps：
(3-1) prepares the DNA profiling of single stranded PCR products：Step (2) is obtained into pcr amplification product and Streptavidin bag
The magnetic bead reaction wrapped up in, the PCR primer DNA expanded by the uncoded primer of biotin modification by 5 ' ends is fixed to the magnetic bead
On, obtain the single-stranded DNA profiling of single stranded PCR products；The PCR for wherein being expanded by the uncoded primer of biotin modification by 5 ' ends is produced
Thing DNA is that another is not encoded 5 ' ends of primer by biotin modification in PCR primer, and its amplified production is 5 ' ends and is given birth to
The DNA of thing element modification；
(3-2) sequencing primer hybridizes：(sequencing primer is sequence known to burnt sequencing primer, i.e., a section to the sequencing primer that will synthesize
Row, and one section of complete complementary in the DNA profiling sequence being sequenced, herein its sequence with encode it is general in primer sequence
Regional sequence is identical) combined with the DNA profiling of single stranded PCR products；
(3-3) designs nucleotides addition sequence and carries out burnt sequencing analysis according to Coding region sequence.
Further, the information of the encoding base in step (3-3) described Coding region sequence is to add single ddNTP to come
Realize decoding；Path base information is realized by adding single dNTP to be sequenced, when a coding for Coding region sequence
After the completion of base is sequenced, the subsequent alkaline gene sequencing primer of this Coding region sequence is closed, not in offer sequencing information,
According to nucleotides add to obtain order, decode successively, when path base is without sequencing information, show next code regional sequence without
PCR primer, stops sequencing reaction.
Further, step (4) is described surveys sequence results according to Jiao, if the ddNTP for adding has obvious sequencing signal,
The PCR primer for showing the coding primer sequence is exist, and shows the characteristic DNA sequence containing the PCR primer in sample；If fruit
Without obvious sequencing signal, then the PCR primer for showing the coding primer sequence is non-existent to the ddNTP of addition, shows sample
In do not contain the characteristic DNA sequence of the PCR primer.
Burnt sequencing analysis method of the present invention based on coding multiple PCR products, the coding primer by particular design is to compile
Code multiple PCR primer, is expanded by coding primer and the increasing of uncoded primer obtains PCR primer.Given birth at 5 ' ends of wherein uncoded primer
Thing element modification, its amplification chain is 5 ' ends by the DNA of biotin modification and amplification chain is complete with coding primer sequence comprising one section
Complementary series fragment, finally encodes the sequence area of primer using burnt sequencing sequentially determining multiplex PCR, determines fgs encoder primer
Whether expand, judge whether to analyze object.Because the information that each coding is respectively provided with uniqueness and decoding also comes from spy
Fixed unique PCR primer.Therefore, sequencing result determines that coding whether there is, and then judges whether the corresponding PCR primer of coding deposits
.Burnt sequencing (decoding) analysis method of the coding multiple PCR products can one by one decode true successively to the PCR primer for encoding
It is fixed.PCR primer correspondence because multiple PCR products are mixed therefore different is unique to be encoded and can not mutually do
Disturb.
Beneficial effect：The burnt sequencing reaction that the inventive method passes through one-time continuous, it is possible to achieve multiplex PCR is possible to produce
The qualitative and quantitative analysis of thing, reduces the workload of detection, shortens the operating time, is especially suitable for the micro- of the industries such as food, health
Biological quick detection and examination.The method of the present invention can shorten detection cycle, to some malignant event (acute infectious diseases
Deng) truth finds out faster, can more gain time correct reply, and the negative effect to society is fewer；While reduction workload,
Reduce testing cost, if implementing one-time detection for each possible detection object, each sample need to implement tens times,
The detection of even up to a hundred times, undoubtedly considerably increases workload, also increases the expense of detection；And multiple detection objects are real parallel
Detection is applied to significantly reduce workload, reduce testing cost.
Compared with prior art, the invention has the advantages that：
1st, the method for the present invention can be implemented to analyze simultaneously to the mix products of multiplex PCR.
2nd, the present invention is compared with conventional gel electrophoretic analysis multiple PCR products, in multiplex PCR different product it is relatively long
Degree is without limitation.
3rd, it is of the invention with it is existingMultiple analysis kit is compared, and can provide multiplex PCR quantitative point
Analyse and operation is more easy, the instrument price for using is more cheap, be more suitable for the application such as clinical diagnosis.
Brief description of the drawings
Fig. 1 is that a kind of composition of the burnt sequencing analysis method coding primer based on coding multiple PCR products is illustrated；
Fig. 2 is that a kind of burnt sequencing analysis method coding primer based on coding multiple PCR products participates in PCR reactions and PCR
The decoding of product is illustrated；
Fig. 3 is Coding region sequence in a kind of burnt sequencing analysis method coding primer based on coding multiple PCR products
A kind of schematic diagram of coding method；
Fig. 4 is a kind of burnt sequencing decoding of burnt sequencing analysis method based on coding multiple PCR products according to Fig. 3 coded systems
Collection of illustrative plates is illustrated；
Fig. 5 is the burnt sequencing row result schematic diagram of embodiment 2.
Specific embodiment
Below in conjunction with drawings and Examples, the invention will be further described.
Embodiment 1
Burnt sequencing analysis method based on coding multiple PCR products：
(1) genomic DNA of sample is extracted；
(2) it is template by encoding the multiplexed PCR amplification of primer with the genomic DNA of sample, obtains and contain mesh to be analyzed
Fragment PCR primer.
Coding primer refers to that expanded in the two of purpose fragment to be analyzed PCR primers is encoded primer, and
One coding one PCR primer of correspondence.As shown in figure 1, coding primer is made up of three subregion sequences, i.e. general areas sequence
1st, Coding region sequence 2 and specific hybridization regional sequence 3.Each different coding primer has specific different specific hybridization area
Domain sequence 3 and Coding region sequence 2, all coding primers all have identical general areas sequence 1.Specific hybridization regional sequence
3 is purpose fragment to be analyzed, be characterize one section of characteristic sequence of object (such as certain microorganism) to be analyzed, with uniqueness,
As specific PCR primer；Coding region sequence 2 is used to mark to determine PCR primer；General areas sequence 1 is used to encode to be surveyed by burnt
Sequence analyzes the sequencing primer hybridising region of (decoding) Coding region sequence；Coding region sequence 2 by 1 encoding base and several
Path base is constituted, wherein last base of 3 ' ends is used to encode in Coding region sequence 2, referred to as encoding base,
Sequence remaining base is referred to as path base.Coding primer is at least 2, typically from 10~20, and each coding primer sequence
Row are respectively provided with uniqueness.
Another primer is not encoded in constituting two PCR primers, and 5 ' ends of the uncoded primer are by biotin modification；Institute
Stating uncoded primer can constitute one the two of the PCR primer of potential purpose fragment PCR primer of amplification with a coding primer,
Can also be used as another public primer of multiple coding primer.If there is specific pcr template sequence in sample genome
Be amplified, then uncoded primer PCR product include and coding primer sequence complete complementary one section of sequence, and PCR primer length
No more than 500bp.
(3) step (2) acquisition PCR primer is prepared into single stranded PCR products, the coding of burnt sequencing analysis single stranded PCR products
Primer (middle coding region) sequence information, concretely comprises the following steps：
(3-1) prepares the DNA profiling of single stranded PCR products：Step (2) is obtained into pcr amplification product and Streptavidin bag
The magnetic bead reaction wrapped up in, the DNA expanded by the uncoded primer of biotin modification by 5 ' ends is fixed on the magnetic bead, is obtained
The DNA profiling of single stranded PCR products；
(3-2) sequencing primer hybridizes：The sequencing primer for synthesizing is combined with the DNA profiling of single stranded PCR products；Sequencing primer
It is burnt sequencing primer, its sequence is identical with general areas sequence in coding primer；
(3-3) designs nucleotides addition sequence and carries out burnt sequencing analysis according to Coding region sequence 2.Coding region sequence 2
In the information of encoding base be to add single ddNTP to realize decoding；Path base information is by adding single dNTP
Sequencing realize, after the completion of the encoding base of a Coding region sequence 2 is sequenced, this Coding region sequence 2 it is follow-up
Alkali gene sequencing primer is closed, and is not providing sequencing information, and order is added to obtain according to nucleotides, is decoded successively, when path alkali
When base is without sequencing information, show that next code regional sequence 2, without PCR primer, stops sequencing reaction.
(4) sequence results are surveyed according to Jiao, it is determined that encode corresponding PCR primer whether there is, realizes the PCR primer feature
The detection of DNA sequence dna.If the ddNTP for adding has obvious sequencing signal, the PCR primer for showing the coding primer sequence is to deposit
, show the characteristic DNA sequence containing the PCR primer in sample；If the ddNTP that fruit adds is without obvious sequencing signal,
The PCR primer for showing the coding primer sequence is non-existent, shows not containing the characteristic DNA sequence of the PCR primer in sample.
As shown in Fig. 24 is coding primer, 5 is genomic DNA, and 6 is another PCR primer, wherein 5 ' are repaiied by biotin 7
Decorations, 8 is biotin modification extended chain, and 9 is the magnetic bead of Streptavidin parcel, and 10 is burnt sequencing primer, its sequence and coding
General areas sequence is identical in primer.A) needed containing coding primer 4, the Mdification primer 6 of biotin 7, genome 5, and PCR
Other compositions (including polymerase, cushioning liquid, the Mg for wanting2+) reacted by PCR, obtain prolonging containing one 5 ' end biotin modification
Stretch the PCR primer of chain 8, wherein sequence of the 3 ' of extended chain 8 comprising one section with coding primer complete complementary；B) PCR is expanded and is produced
Thing is reacted with the magnetic bead of Streptavidin parcel, and the decorations extended chain 8 of biotin modification is fixed on magnetic bead 9, obtains single stranded DNA mould
Plate；C) sequencing by hybridization primer and sequencing, burnt sequencing decoding coding is carried out according to Coding region sequence design nucleotides addition sequence
Information.Due to sequence of the extended chain 5 ' comprising one section with coding primer complete complementary, therefore, the nucleotides that sequencing decoding is added is suitable
Sequence is exactly to be sequentially added according to the order of coding region, and simply encoding base is determined and adds corresponding ddNTP, and road sign base is determined
Add corresponding dNTP.
As shown in figure 3, Coding region sequence is made up of 1 encoding base and several road sign bases, wherein encoding base
It is 1 (bold-faced letter in figure) that remaining is path base (non-black-body letter in figure).In figure 3, coding 1 regional sequence be 5 '-
AT-3 ', wherein A are road sign base, and T is encoding base, its coding primer 1；8 regional sequences are encoded for 5 '-ACACG-3 ', wherein
A, C, A, C are road sign base, and G is encoding base, its coding primer 8；Other the like.It can be found that this coding is produced
Coded number n be with the relation of coding primer sequence base number N：N=2 (N-1).
Decoding does not cause the influence to subsequent analysis by not continuing to extend nucleotides.Decoding is according to the single ddNTP of addition
One sequencing reaction decodes a coding (base), according to the order of coding sequentially add corresponding single ddNTP sequencings decoding,
Until completing all decodings without sequencing information, as shown in figure 4, being firstly added dATP carries out sequencing reaction, its sequencing intensity letter
The total amount of all different PCR primers of breath reaction, can be as quantitative analysis benchmark (100%).Then successively, according to volume
Code regional sequence design nucleotides addition sequence carries out burnt sequencing decoding coding information.It should be noted that encoding base is determined adding
Enter corresponding ddNTP, road sign base is determined and adds corresponding dNTP.
Decoding coding 1 is that addition ddTTP carries out sequencing reaction：If the sequencing strength information more than background, then show
The PCR primer of the coding 1 is present, and its content is this sequencing information intensity/the first road sign base sequencing information intensity；Such as nothing
More than the sequencing strength information of background, then show that the PCR primer of the coding does not exist.Similarly, adding ddGTP sequencings can solve
Code coding 2.It is obvious that after the completion of the decoding of coding 1,2, the PCR primer for no matter encoding 1,2 whether there is, both codings
PCR sequences will not contribute sequencing information to follow-up sequencing reaction：If the PCR primer of coding 1,2 does not exist, nature will not
Produce any sequencing information；If the PCR primer of coding 1,2 is present, due to ddTTP, ddGTP for adding, all and coding 1,2
PCR primer hybridization sequencing primer be closed, will not continue to occur extension, will not naturally also produce again sequencing believe
Number.After 1,2 completion decoding is encoded, dCTP is added to determine a road sign base；Then being separately added into ddTTP, ddGTP again can be with
Decoding coding 3,4.So, under the guide of road sign base, more codings just can be decoded and calculate phase by information strength
The content answered.When the sequencing information of road sign base and the consistent sequencing intensity of background, show the PCR primer army headquaters of next code
In the presence of completing all codings and be decoded.
Embodiment 2
A kind of HPV classifying methods based on coding multiple PCR products：
(1) opening of the cervix is placed in special Uterine neck bush, gently rubbing Uterine neck bush makes its 5 circle that turn clockwise, and slowly takes out palace
Neck brush, puts it into the probe tube for indicating patient code, completes sample collection, using DNA in kit extraction sample；
(2) it is template by encoding the multiplexed PCR amplification of primer with the DNA of sample, obtains and contain purpose fragment to be analyzed
PCR primer：Using 15 kinds of HPV genotype as object to be analyzed, every kind of coding primer encodes a kind of HPV genotype, including three
Part：General areas sequence, Coding region sequence and specific hybridization regional sequence, wherein specific hybridization regional sequence are to be analyzed
The purpose fragment of HPV genotype, be characterize one section of characteristic sequence of specific HPV genotype, with uniqueness, and be encoded region
Sequential coding.The plain non-coding primer SEQ ID NO 16 of every kind of coding primer and 5 ' modified biologicals (5 '-
GAAAAATAAACTGTAAATCA-3 ') constitute PCR two amplimers.
The specific coding mode for encoding primer is as follows：
The PCR amplification system of 50 μ L is included：200ng sample DNAs, every kind of coding primer of 0.8mM dNTPs, 10pmol and
Non-coding primer SEQ ID NO 16 (5 '-GAAAAATAAACTGTAAATCA-3 ') of 0.8 μM of 5 ' modified biologicals element, 1U
Taq archaeal dna polymerases, 1 × amplification buffer, 2.5mM MgCl2.Amplification condition：94 DEG C of predegenerations 10 minutes, 45 thermal cycles：
945 DEG C of 30 seconds~52 DEG C of denaturation, 45 seconds~72 DEG C of annealing extend 30 seconds, and last 72 DEG C extend 7 minutes.
(3) by PCR primer and the magnetic bead reaction of streptavidin modification, the DNA templates of single stranded PCR products are obtained, will be surveyed
The DNA of the single-stranded PCR that sequence primer (i.e. with the identical sequence in universal sequence region in coding primer sequence) is fixed with magnetic bead
Template is reacted 5 minutes at 80 DEG C, then naturally cools to room temperature.Nucleotides addition sequence is designed according to Coding region sequence to enter
Row Jiao sequencing analysis, as shown in figure 4, according to dATP, ddTTP, ddGTP, dCTP, ddTTP, ddGTP, dATP, ddTTP,
DdGTP, dCTP, ddTTP, ddGTP, dATP, ddTTP, ddGTP, dCTP, ddTTP, ddGTP order sequentially add nucleotides and enter
Row sequencing, decoding coding, determines the HPV partings of sample.Abscissa is (sequencing) signal intensity in Fig. 4, and ordinate is (sequencing)
The sequencing reaction liquid or nucleotides of addition, wherein E are buffering reaction solution, and S is enzyme reaction solution, and black font is corresponding
DdNTP, non-black font is corresponding dNTP.The sequencing information of ddNTP decodes a coding and whether there is, dNTP sequencing informations
Represent that path whether there is, wherein the sequencing strength information of first path base reacts the total amount of all different PCR primers, make
It is a benchmark (100%) of quantitative analysis
(4) sequence results are surveyed according to Jiao, it is determined that encode corresponding PCR primer whether there is, realizes the PCR primer feature
The detection of DNA sequence dna：It is as shown in Figure 5 that Jiao surveys sequence results.When dATP is added, there is obvious sequencing peak, show PCR primer
In the presence of；When adding ddTTP to carry out sequencing reaction decoding coding 1HPV16, sequencing intensity is suitable with background for it, show coding 1
PCR primer does not exist；Similarly, add ddGTP sequencing can decode coding 2HPV 66, its sequencing intensity it is suitable with also background,
Show that the PCR primer of coding 2 does not exist yet；When adding dCTP to carry out sequencing reaction, there is obvious sequencing peak, show that PCR is produced
Thing is present；When adding ddTTP to carry out sequencing reaction decoding coding 3HPV 59, sequencing intensity is suitable with background for it, show coding 3
PCR primer do not exist；Adding ddGTP sequencings can decode coding 4, and its sequencing intensity is significantly stronger than background, shows coding 4
PCR primer is present, that is, show to contain the viruses of 4 corresponding characteristic sequence HPV of coding 56 in sample；It is sequenced when dATP is added
Reaction, sequencing intensity is suitable with background for it, show that follow-up PCR primer is entirely absent, and completes being decoded for all codings.Cause
This, sample only detects the viruses of characteristic sequence HPV 56 that coding primer sequence 4 is represented from 15 detection objects to be analyzed,
And other 14 kinds do not detect then.
SEQUENCE LISTING
<110>Southeast China University
<120>A kind of burnt sequencing analysis method based on coding multiple PCR products
<130> 2017
<160> 16
<170> PatentIn version 3.3
<210> 1
<211> 45
<212> DNA
<213> HPV 16
<400> 1
ggaaggtgct cctctgcact cattgtcatt atgtgctgcc atatc 45
<210> 2
<211> 46
<212> DNA
<213> HPV 66
<400> 2
ggaaggtgct cctctgcact cagactatta atgcagctaa aagcac 46
<210> 3
<211> 46
<212> DNA
<213> HPV 59
<400> 3
ggaaggtgct cctctgcact cacttgtgct ctactactct ctattc 46
<210> 4
<211> 45
<212> DNA
<213> HPV 56
<400> 4
ggaaggtgct cctctgcact cacgtactgc tacagaacag ttaac 45
<210> 5
<211> 46
<212> DNA
<213> HPV 33
<400> 5
ggaaggtgct cctctgcact cacattatgc acacaagtaa ctagtg 46
<210> 6
<211> 46
<212> DNA
<213> HPV 39
<400> 6
ggaaggtgct cctctgcact cacagagagt cttccatacc ttctac 46
<210> 7
<211> 46
<212> DNA
<213> HPV 35
<400> 7
ggaaggtgct cctctgcact cacacttgtt ctgctgtgtc ttctag 46
<210> 8
<211> 46
<212> DNA
<213> HPV 45
<400> 8
ggaaggtgct cctctgcact cacacgccaa gtacatatga ccctac 46
<210> 9
<211> 47
<212> DNA
<213> HPV 51
<400> 9
actgccactg ctgcggtttc actctaggaa ggtgctcctc tgcactc 47
<210> 10
<211> 47
<212> DNA
<213> HPV 68
<400> 10
ggaaggtgct cctctgcact cacacagact actgaatcag ctgtacc 47
<210> 11
<211> 48
<212> DNA
<213> HPV 82
<400> 11
ggaaggtgct cctctgcact cacacacttg ttactccatc tgttgcac 48
<210> 12
<211> 48
<212> DNA
<213> HPV 58
<400> 12
ggaaggtgct cctctgcact cacacacgac tgaagtaact aaggaagg 48
<210> 13
<211> 48
<212> DNA
<213> HPV 18
<400> 13
ggaaggtgct cctctgcact cacacacata cagtctcctg tacctggg 48
<210> 14
<211> 48
<212> DNA
<213> HPV 31
<400> 14
ggaaggtgct cctctgcact cacacacagc aattgcaaac agtgatac 48
<210> 15
<211> 48
<212> DNA
<213> HPV 52
<400> 15
ggaaggtgct cctctgcact cacacacact tgactttatg tgctgagg 48
<210> 16
<211> 20
<212> DNA
<213>It is artificial synthesized
<400> 16
gaaaaataaa ctgtaaatca 20
Claims (10)
1. it is a kind of based on the burnt sequencing analysis method for encoding multiple PCR products, it is characterised in that to comprise the following steps：
(1) genomic DNA of sample is extracted；
(2) it is template by encoding the multiplexed PCR amplification of primer with the genomic DNA of sample, obtains and contain purpose to be analyzed
The PCR primer of fragment；
(3) step (2) acquisition PCR primer is prepared into single stranded PCR products, the coding primer of burnt sequencing analysis single stranded PCR products
The sequence information of middle coding region；
(4) sequence results are surveyed according to Jiao, it is determined that encode corresponding PCR primer whether there is, realizes the PCR primer characteristic DNA sequence
The detection of row.
2. according to right wants 1 based on coding multiple PCR products burnt sequencing analysis method, it is characterised in that step (2)
It is described coding primer refer to amplification one the two of purpose fragment to be analyzed PCR primer in one be coded of primer, and one
Coding one PCR primer of correspondence；Another PCR primer is uncoded primer.
3. it is according to claim 1 and 2 based on the burnt sequencing analysis method for encoding multiple PCR products, it is characterised in that institute
State coding primer to be made up of three subregion sequences, i.e. specific hybridization regional sequence, Coding region sequence and general areas sequence；
The specific hybridization regional sequence is used as specific PCR primer；The Coding region sequence is used to mark to determine PCR primer；It is described logical
It is used for the sequencing primer hybridising region of burnt sequencing analysis Coding region sequence with regional sequence.
4. it is according to claim 3 based on the burnt sequencing analysis method for encoding multiple PCR products, it is characterised in that described
Coding region sequence is made up of 1 encoding base and several path bases, wherein 3 ' ends are most in Coding region sequence
Latter base is used to encode, referred to as encoding base, and sequence remaining base is referred to as path base.
5. it is according to claim 3 based on the burnt sequencing analysis method for encoding multiple PCR products, it is characterised in that described
The specific hybridization regional sequence for encoding primer is purpose fragment to be analyzed, is one section of characteristic sequence, the tool for characterizing object to be analyzed
There is uniqueness.
6. it is according to claim 1 and 2 based on the burnt sequencing analysis method for encoding multiple PCR products, it is characterised in that institute
The purpose fragment for stating a coding primer represents an object to be analyzed, and coding primer is at least 2, i.e., at least to be analyzed right
As being 2, multiplex PCR is constituted, coding primer is preferably 10~20.
7. it is according to claim 2 based on the burnt sequencing analysis method for encoding multiple PCR products, it is characterised in that described
5 ' ends of another uncoded primer are by biotin modification in two PCR primers；The uncoded primer can be with a coding
Primer constitutes one the two of purpose fragment to be analyzed PCR primer of amplification, it is also possible to used as multiple coding public another of primers
Individual primer.
8. according to right wants 1 based on coding multiple PCR products burnt sequencing analysis method, it is characterised in that step (3)
Concretely comprise the following steps：
(3-1) prepares the DNA profiling of single stranded PCR products：Step (2) is obtained into pcr amplification product with Streptavidin parcel
Magnetic bead reacts, and is fixed on the magnetic bead by the uncoded primer amplification PCR primer DNA of biotin modification by 5 ' ends, obtains
The DNA profiling of single stranded PCR products；
(3-2) sequencing primer hybridizes：The sequencing primer for synthesizing is combined with the DNA profiling of single stranded PCR products；The sequencing primer
It is burnt sequencing primer, its sequence is identical with general areas sequence in coding primer；
(3-3) designs nucleotides addition sequence and carries out burnt sequencing analysis according to Coding region sequence.
9. according to right wants 8 based on coding multiple PCR products burnt sequencing analysis method, it is characterised in that step (3-
3) information of the encoding base in the Coding region sequence is to add single ddNTP to realize decoding；Path base information
It is to be realized by adding single dNTP to be sequenced, after the completion of the encoding base of a Coding region sequence is sequenced, this volume
The subsequent alkaline gene sequencing primer of code regional sequence is closed, and is not providing sequencing information, and order is added to obtain according to nucleotides, according to
Secondary decoding, when path base is without sequencing information, shows that next code regional sequence, without PCR primer, stops sequencing reaction.
10. according to right wants 1 based on coding multiple PCR products burnt sequencing analysis method, it is characterised in that step
(4) it is described that sequence results are surveyed according to Jiao, if the ddNTP for adding has obvious sequencing signal, show the coding primer sequence
PCR primer is exist, and shows the characteristic DNA sequence containing the PCR primer in sample；If the ddNTP that fruit adds is without obvious
Sequencing signal, then the PCR primer for showing the coding primer sequence is non-existent, shows not containing the PCR primer in sample
Characteristic DNA sequence.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104404160A (en) * | 2014-12-09 | 2015-03-11 | 南京大学 | MIT (Mitochondrion) primer design method and method for constructing planktonic animal barcode database by utilization of high-throughput sequencing |
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2017
- 2017-04-17 CN CN201710249100.1A patent/CN106929589A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104404160A (en) * | 2014-12-09 | 2015-03-11 | 南京大学 | MIT (Mitochondrion) primer design method and method for constructing planktonic animal barcode database by utilization of high-throughput sequencing |
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| XIAODAN ZHANG ET AL.: "Construction of 3-Plex Barcodes for Differential Gene Expression Analysis with Pyrosequencing", 《ADVANCES AND CLINICAL PRACTICE IN PYROSEQUENCING》 * |
| ZHIYAO CHEN ET AL.: "Development of Pyrosequencing-Based Multiplex Bioassay by Designing Barcodes Encoded with Artificially Designed Sequences", 《ADVANCES AND CLINICAL PRACTICE IN PYROSEQUENCING》 * |
| 曹冬梅 等: "PCR-焦磷酸测序法快速鉴定食品中肠炎沙门菌", 《大连工业大学学报》 * |
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