CN109652518A - A kind of kit detecting minimal residual disease MRD - Google Patents

A kind of kit detecting minimal residual disease MRD Download PDF

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CN109652518A
CN109652518A CN201811550133.0A CN201811550133A CN109652518A CN 109652518 A CN109652518 A CN 109652518A CN 201811550133 A CN201811550133 A CN 201811550133A CN 109652518 A CN109652518 A CN 109652518A
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孙涛
余莹莹
张天骄
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Hangzhou Aimuanthene Biotechnology Co Ltd
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Abstract

It is hematologic cancers (such as T/B system leukaemia the present invention provides a kind of detection leaching based on high-flux sequence research and development, lymthoma, myeloma minimal residual disease) kit, the kit includes detection IGH (VDJ), IGH (DJ), IGK, IGL, TCR β, TCR γ, TCR δ, BCL1/IGH, BCL2/IGH's has UMB (Unique Molecular Barcode, unimolecule label) multiple PCR primer group, House Keeping gene PCR primer pair, Multiplex PCR Mix (2X), internal reference DNA, Nuclease-Free Water, Elution Buffer, and high pass Primer and sequence label needed for measuring sequence.Using the kit, the cancer cell of new mutation can be found while detecting cancer cell, and apply high throughput sequencing technologies combination bioinformatic analysis, the detection sensitivity of MRD can at least detect a cancer cell in 1,000,000 cells, i.e., 10‑6(0.0001%), it not only can reach minimal residual disease testing goal with quantitative analysis cancer cell number, and the sequence read mistake generated in series jump caused by the base mispairing of PCR amplification generation and library sequencing procedure can be corrected.

Description

A kind of kit detecting minimal residual disease MRD
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of kit for detecting minimal residual disease MRD.
Background technique
Leaching is that hematologic cancers mainly include T/B lymphocytic leukemia, lymthoma and Huppert's disease, and small residual Disease (Minimal Residual Disease, MRD) is stayed to refer to that leukaemia/lymthoma/patients with malignant myeloma is alleviated through inductive treatment Afterwards, the state of a small amount of cancer cell is still remained in vivo, may finally cause palindromia.Leukaemia can be divided into the white blood of lymphocyte Disease, myelocytic leukemia, cell mixing leukaemia are a kind of candidate stem cell malignant clone diseases.Clonal leukaemia is thin Born of the same parents' mechanism largely proliferation accumulation in marrow and other hematopoietic tissues because proliferation out of control, dysdifferentiation, apoptosis are obstructed etc., and soak Moisten other non-hematopoietic tissues and organ, while inhibiting normal hematopoiesis function.Lymthoma is initiated by the pernicious of lymphohematological Tumour can be divided into B cell, T cell, NK cell lymphoma according to different lymphocytes origin.Huppert's disease is a kind of gram The malignant tumour of grand property thick liquid cell abnormality proliferation, tumour cell is originating from the thick liquid cell in marrow, and thick liquid cell is that B lymph is thin Born of the same parents develop the cell to final function phases.
With continuously improving for the technologies such as chemotherapy, selectively targeted treatment, hematopoietic stem cell transplantation (HSCT) treatment, blood The therapeutic effect of cancer is even up to hematology complete incidence graph (hematologic complete remission, HCR), but It obtains HCR and is not sufficient to guarantee patient's Long-term disease-free survival, therefore recurrence is still the difficult point for perplexing cancer and curing, and studies carefully its original Because mainly related with hematologic cancer cell minimal residual disease (MRD).
Recent study shows that hematologic cancers recurrence is closely related with MRD, and MRD raising can indicate hematologic cancers in advance Recurrence comprehensively.Therefore, hematologic cancers patient is carried out using high sensitivity, high specificity and reliable and stable experimental method regular MRD detection to assessment morbid state, judges that curative effect, prediction recurrence, guiding treatment have important clinical meaning.Hematologic cancers Therapeutic scheme and novel effective drug cooperation, patient disease alleviation doubles, MRD monitoring time point and detection spirit Sensitivity is particularly important for recurrence prediction.
A large amount of V (variable region) on B/T cytogene seat, D (variable region), J (bonding pad) genetic fragment B/T cell by Diversity recombination can be generated in the forming process of body.The recombination of this V-D-J gene imparts each B/T cell oneself uniqueness Cell receptor (BCR/TCR) so that the sequence of each BCR/TCR can effectively become B/T cell clone Specific biological marker.Therefore BCR/TCR gene order composition is sequenced, can be very good to position each B/T cell, Including the B/T cell of canceration, and MRD detection sensitivity is up at least up to 10-6(0.0001%).
Since the maximum feature of BCR/TCR gene is the random recombination of V, D, J genetic fragment, according to various known V, D, J The multi-primers of the conservative region design of gene expand different receptor chains, then add sequencing label.But in cancer patient, cancer is thin The gene mutation of born of the same parents be it is very common, if the BCR/TCR of cancer cell produces mutation, the primer of known array just has can Gene after capable of can not identifying mutation is easy for causing false negative for testing result.
In the prior art, document " Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombination in suspect lymphoproliferations:Report of the BIOMED-2 Concerted Action BMH4-CT98-3936 " in devise 3 VH-JH, 2 DH-JH, 2 IGK, an IGL, 3 TCRB, 2 TCRG, 1 TCRD, 3 BCL1-IGH, a BCL2-IGH have 18 multi-PRC reactions altogether, relate to 107 This set primer is finally named as BIOMED2 by different primers.But primer does not cover all V, J genetic fragments, is easy to produce Multiple PCR primer Preference is not directly suitable for high-flux sequence, and can not carry out PCR and sequencing amplification mispairing reparation. Document " Deep-sequencing approach for minimal residual disease detection in acute Lymphoblastic leukemia " in using internal reference establish the computation model of cancer cell.
The method of detection minimal residual disease (MRD) includes that multi-parameter flow cytomery (FC), polymerase chain are anti-at present Answer (PCR), fusion detection real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), allele oligonucleotide hybridization method (AS0-PCR) and (IG/TR NGS) etc. is sequenced in high-throughput two generations.The most commonly used is the fluidic cells based on molecular immunology Instrument art (Flow Cytometry), but its sensitivity is only up to 10-4The order of magnitude (0.01%), and this method judges MRD result Accuracy depend greatly on the experiment condition in each laboratory and the personal experience of operator, standardization level It is low, and some researches show that the influences in chemotherapy process due to chemotherapeutics, and the immunophenotype of cancer cell to be made to change, i.e., " immunological drift " phenomenon can influence the reliability and accuracy of MRD testing result.The method of polymerase chain reaction (PCR) can not Quantitatively, the case where can only see the gene rearrangement of IG/TCR.Fusion real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) Detection method can be only applied to that the source of leukaemia cell cannot be prompted there are the acute leukemia of fusion exception.Equipotential The application of gene oligonucleotide hybridization assay (AS0-PCR) is relatively prevalent in Europe, and this method needs fixed according to each patient A set of primer is made, non-specific amplification is easy to appear, experiment condition and operating technology are more demanding, time-consuming consumption manpower, and cancer Cellular nucleic acid, which mutates, will result in the false negative of MRD detection.
Summary of the invention
In order to solve the above technical problems, The present invention provides a kind of kit for detecting minimal residual disease MRD, the reagents Box can find the cancer cell of new mutation while can detecte cancer cell；Meanwhile the kit uses high throughput sequencing technologies knot Close bioinformatic analysis, the detection sensitivity of MRD in 1,000,000 cells up at least detecting a cancer cell, i.e., 10-6 (0.0001%).
On the one hand, the present invention provides it is a kind of detect minimal residual disease MRD kit, including build library kit and Illuminaindex kit, wherein it is described build library kit include detect respectively IgH (VDJ), IgH (DJ), IgK, IgL, TRB, TRG, TRD, BCL1/IGH, BCL2/IGH with UMB (Unique Molecular Barcode, unimolecule label) Multiple PCR primer group, House Keeping gene PCR primer and internal reference DNA.
Further, wherein it is described detection IgH PCR primer in, upstream primer sequence as shown in SEQ ID NO:1-16, Downstream primer sequence is as shown in SEQ ID NO:24；In the primer for wherein detecting IgH (DJ), upstream primer sequence such as SEQ ID Shown in NO:17-23, downstream primer sequence is as shown in SEQ ID NO:24；In the primer for wherein detecting IgK, upstream primer sequence As shown in SEQ ID NO:25-34, downstream primer sequence is as shown in SEQ ID NO:35-37；In the primer for wherein detecting IgL, Upstream primer sequence is as shown in SEQ ID NO:38-54, and downstream primer sequence is as shown in SEQ ID NO:55-57；Wherein detect In the primer of TRB, upstream primer sequence is as shown in SEQ ID NO:58-88, downstream primer sequence such as SEQ ID NO:89-100 It is shown；In the primer for wherein detecting TRG, upstream primer sequence is as shown in SEQ ID NO:101-106, and downstream primer sequence is such as Shown in SEQ ID NO:107-109；In the primer for wherein detecting TRD, upstream primer sequence such as SEQ ID NO:110-112 institute Show, downstream primer sequence is as shown in SEQ ID NO:113-116；Detect the primer pair of BCL1/IGH, upstream primer sequence such as SEQ Shown in ID NO:117, downstream primer sequence is as shown in SEQ ID NO:118；Detect the primer pair of BCL2/IGH, upstream primer sequence Column are as shown in SEQ ID NO:119-127, and downstream primer sequence is as shown in SEQ ID NO:118；Wherein N:A or T or C or G；R: A or G；W:A or T；Y:C or T；S:C or G；B:T or C or G.
Further, wherein to build further include 2 in the kit of library ×Premix, Multiplex PCR Mix (2X), Nuclease-Free Water, Elution Buffer.
Further, the House Keeping gene primer sequence is SEQ ID NO:250-253.
Further, the internal reference DNA sequence dna is shown in SEQ ID NO:128-226.
Further, wherein including sequencing primer and label in the illumina index kit, the sequencing is drawn The sequence of object and label is shown in SEQ ID NO:227-247, and the sequence of middle and upper reaches sequencing primer and label is SEQ ID Shown in NO:227-238, the sequence of downstream sequencing primer and label is shown in SEQ ID NO:239-247.
Further, the content of each ingredient is different according to the different size of kit in kit.
Table 1 builds the specific ingredient and content of library kit
The specific ingredient and content of 2 illumina index kit of table
On the other hand, the present invention provides the methods that a kind of couple of T, B-cell receptor carry out high-flux sequence, using described Kit, specifically includes the following steps:
(1) sample to be tested is taken；
(2) sample to be tested is expanded using the library kit of building in the kit；
(3) product of step (2) is expanded using the illumina index kit in the kit；
(4) bioinformatic analysis carries out quantitative and qualitative analysis to amplified production.
Optionally, according to target gene (IGH (VDJ), IGH (DJ), IGK, IGL, TCR β, TCR γ, TCR in step (2) δ, BCL1/IGH, BCL2/IGH) it is added in PCR reaction system known to any three corresponding 1%input DNA profiling amounts The internal reference DNA of sequence carries out specific amplification with sample simultaneously.
Optionally, the internal reference DNA sequence dna is shown in SEQ ID NO:128-226.
Optionally, House Keeping gene β-actin is expanded simultaneously in the step (2), the primer sequence of use is such as SEQ ID NO:250-251, or House Keeping gene GAPDH is expanded simultaneously, the primer sequence of use such as SEQ ID NO: 250-251。
Optionally, the sequence of sequencing primer described in step (3) and label be SEQ ID NO:227-247 shown in, wherein Upstream primer sequence is shown in SEQ ID NO:227-238, and downstream primer sequence is shown in SEQ ID NO:239-247.
Optionally, the bioinformatic analysis: by the immune group library fastq file of NGS high-flux sequence, remove low Machine sequencing sequence under quality determines V (D) J gene after comparing with IMGT database, and assembling obtains complete CDR3 sequence, then Being removed according to clone's frequency may polluted sequence.MRD value takes two kinds of calculations.One, using internal reference as quantitative criterion.MRD =(Rc/(Rstd/Nstd)/2)/NTOT, wherein RcIt is the reads number of sequencing gained cancer cell, RstdIt is sequencing gained internalcontrol sequence Reads number, NstdIt is the molecular number for adding internal reference, NTOT=internal reference gross mass/each reference molecules molal weight, NTOT= MTOT/ K, NTOTIt is the quantity for always having core living cells, MTOTIt is the quality i.e. quality of input DNA for always having core living cells, K is every A cell DNA total amount 0.0064ng.Two, using House Keeping gene as quantitative criterion.There are two types of calculation methods: 1) .MRD=NC/NH, wherein NCIt is sequencing gained cancer cell number according to have passed through UMB analysis treated cancer cell number, NHIt is sequencing Gained HouseKeeping gene data have passed through UMB analysis treated total template cell number.2) .MRD=(Rc/(RH/TH)/ 2)/NTOT, wherein RcIt is the reads number of sequencing gained cancer cell, RHIt is the reads of sequencing gained House Keeping gene Number, THIt is the template number of House Keeping gene magnification, NTOT=internal reference gross mass/each reference molecules molal weight, NTOT =MTOT/ K, NTOTIt is the quantity for always having core living cells, MTOTIt is the quality i.e. quality of input DNA for always having core living cells, K is Each cell DNA total amount 0.0064ng.
The technology of the present invention effect:
1. adjusting suitable primer concentration by over-designed region multi-primers, solving the preference of multiplexed PCR amplification Property.Increase independent molecule label (UMB) of the random sequence as each template in design of primers, every primer sequence is respectively not It is identical, not only quantitatively each DNA molecular can also can be really carried out with error correction.
2. can not only track the sequence as diagnostic flag using this kit, while emerging cancer can also be detected Cell clone sequence, these may imply the progress or recurrence of disease.MRD detection in high sensitivity can occur in clinical symptoms Preceding prediction recurrence, and therapeutic effect is assessed, doctor can be helped early stage to take therapeutic intervention to patient more in time.
3. the pairing of the V and J of complete BCR/TCR, this synthesis are imitated in kit using the internal reference of a set of synthesis Sequence is all highly stable in each detection process, and quantifies to detect the whole karyocytes for including in sample by internal reference (inputDNA).This method is vital to the accuracy for ensuring clinical MRD result.
4. this method House Keeping gene stable using expression quantity, this gene are non-in each detection process Often stablize, can quantify to detect the template number for participating in reaction in sample after analyzing by UMB and the shape before PCR amplification can be restored State.This method is to correct quantitatively MRD the result is that vital.
5. the accuracy that this method ensures clinical sample MRD report in conjunction with exclusive bioinformatic analysis.Correct reason " denominator " used in MRD testing result is solved for explaining that MRD tumor load is most important.The testing result of MRD is cancer cell Number/all has core viable count, then denominator " all to have core living cells " number just will affect the result of final MRD.For example, Cancer cell is " 137 ", is if total cell detects background only in accordance with 10000 B cell BCR sequencing results, this means that MRD value is 137/10000, this is clinically to recur；If with 1,000,000 total cells (all mononuclearcells) for background Detection, then MRD value is 137/10000000, then clinically thinking not recur.Other MRD testing result uses It is the percentage of B cell or T cell, then there is MRD result inaccuracy for the patient of lymphocyte depletion after treatment.
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Detailed description of the invention
The process of Fig. 1 immune group high-flux sequence detection minimal residual disease method.
The construction method in the library Fig. 2
2100 quality inspection of Fig. 3 as a result, on abscissa peak corresponding to 15bp and 1500bp respectively represent low marker and Upmarker, multiple peaks, that is, library size in this range.
Two kinds of calculation methods of Fig. 4 MRD
Fig. 5 bioinformatics treated result example.
Result example of the Fig. 6 after bioinformatics and visualization processing, wherein each circle represents a clone, often The size of a circle indicates the frequency size of the clone, and frequency is bigger, and circle is bigger.
Preceding 10 kinds of clones ranking results example of the Clonal testing result high frequency of Fig. 7 cancer cell
Cancer cell clonal evolution example in Fig. 8 minimal residual disease trace detection result
Specific embodiment
The present invention is further illustrated with embodiment below, but the present invention is not intended to be limited thereto.It is not infused in the following example The experimental method of bright actual conditions, usually according to normal condition, or according to the normal condition proposed by manufacturer.
Embodiment 1: the acquisition of sample genome
1. can be divided into according to pretherapy and post-treatment and sample specificity:
Sample of bone marrow 100uL, 200uL, 300uL, 400uL, 500uL are in EDTA anticoagulant tube before ruling treatment by men；
Rule by men treatment after sample of bone marrow 100uL, 200uL, 300uL, 400uL, 500uL, 600uL, 700uL, 800uL, 900uL, 1mL, 2mL are in EDTA anticoagulant tube；
Human peripheral sample 5mL, 6mL, 7mL, 8mL, 9mL, 10mL is in EDTA anticoagulant tube.
2. having core living cells using separation after red blood cell in erythrocyte cracked liquid lysed sample.
3. extracting genome gDNA after thering is core living cells to carry out cell count gained.
Embodiment 2: multiplexed PCR amplification and library construction
Library kit is built using in the kit, the multipair V base for having UMB is added in multi-PRC reaction system Because of the primer of segment and J genetic fragment, and add any three corresponding 1%input DNA profiling amounts known array it is interior Ginseng DNA or House Keeping gene and sample carry out specific amplification simultaneously.
Primer sets sequence is as shown in table 1
1 multiple PCR primer of table
Primer sequence structure is as shown in Fig. 2, it is SEQ that wherein the Read1 sequence, which is SEQ ID NO:248, Read2 sequence, IDNO:249。
Wherein the sequence of the internal reference DNA is shown in SEQ ID NO:128-226.
Wherein the PCR primer sequence of the amplification House Keeping gene is shown in SEQ ID NO:250-253.
Multiplex PCR system, including 25 μ L, 50 μ L, the following are 25uL systems:
PCR premixed liquid used in multiplex PCR: Multiplex PCR MasterMix (UNG).
PCR reaction caching liquid is mixed to carry out according to following response procedures:
Embodiment 3: high-flux sequence and bioinformatic analysis
The Hiseq system from illumina company is used in method of the invention, Hiseq is a kind of based on unimolecule cluster While synthesis while sequencing technologies, based on proprietary reversible termination chemical principle.The random fragment of DNA is attached to when sequencing Optically transparent glass surface (Flow cell), these DNA fragmentations are after extension and bridge amplification, the shape on Flow cell It is the unimolecule cluster with thousands of parts of same templates at hundreds of millions of clusters (cluster), each cluster.Then using having (Sequencing by is sequenced in synthesis by what invertibity terminated in four kinds of special deoxyribonucleotides of fluorophor Synthesis, SBS) template DNA to be measured is sequenced in technology.
Firstly, the upper machine sequencing primer and mark of illumina high-flux sequence instrument are added at the both ends of PCR1 product respectively Label, while expanding (referring to fig. 2).
The primer that the specified sequencing of Illumina uses and the label applied to data analysis:
Overstriking is label index.
PCR2 reaction buffer is prepared in the following proportions:
PCR2 reaction caching liquid is mixed to carry out according to following response procedures:
The purifying of amplified production: PCR2 product is purified using magnetic bead recovery method
A, 80 μ L AMPure XP Beads of addition enter PCR2 reaction product, mix.
B, at incubation at room temperature 10 minutes.
C, magnetic bead-PCR2 product mixtures test tube is placed after on magnetic frame, waiting all magnetic beads to be adsorbed on magnetic frame, All supernatants are sucked with pipettor, are abandoned.
D, addition 150 μ L, 70% ethyl alcohol hatches 30 seconds on magnetic bead, sucks all supernatants with pipettor, abandons.
E, D step 2 time is repeated.
F, test tube cap is opened, is waited 5 minutes, air-dries to magnetic bead, remains in test tube without any ethyl alcohol.
G, test tube is removed from magnetic frame, and adds 30 μ L and removes nuclease water, blow and beat suspension magnetic bead using pipettor.
H, tube back magnetic frame, after waiting all magnetic beads to be all adsorbed in magnetic frame, supernatant is shifted in new test tube In.
I, the PCR2 product after purifying is just contained in supernatant.
Using Agilent nucleic acid fragment analyzer 2100 to purified product carry out quality inspection, as Fig. 3 be 2100 quality inspections as a result, Peak corresponding to 15bp and 1500bp respectively represents low marker and up marker on abscissa, more in this range A peak, that is, library size (referring to Fig. 3).
It carries out high-flux sequence: resulting DNA library is sequenced by Illumina HiSeq platform, mode is sequenced For PE150, library denaturant concentration is 2nM, and upper machine concentration is 20pM, and passes through bioinformatic analysis high-flux sequence result. One, using internal reference as quantitative criterion.MRD=(Rc/(Rstd/Nstd)/2)/NTOT, wherein RcIt is the reads of sequencing gained cancer cell Number, RstdIt is the reads number of sequencing gained internalcontrol sequence, NstdIt is the molecular number for adding internal reference, NTOT=internal reference gross mass/each Reference molecules molal weight, NTOT=MTOT/ K, NTOTIt is the quantity for always having core living cells, MTOTIt is always to have the quality of core living cells i.e. The quality of input DNA, K are each cell DNA total amount 0.0064ng.Two, using House Keeping gene as quantitative mark It is quasi-.There are two types of calculation methods: 1) .MRD=NC/NH, wherein NCIt is after sequencing gained cancer cell number evidence have passed through UMB analysis processing Cancer cell number, NHIt is total template cell number after sequencing gained House Keeping gene data have passed through at UMB analysis. 2) .MRD=(Rc/(RH/TH)/2)/NTOT, wherein RcIt is the reads number of sequencing gained cancer cell, RHIt is sequencing gained The reads number of HouseKeeping gene, THIt is the template number of House Keeping gene magnification, NTOT=internal reference gross mass/ Each reference molecules molal weight, NTOT=MTOT/ K, NTOTIt is the quantity for always having core living cells, MTOTIt is the matter for always having core living cells Amount is the quality of inputDNA, and K is each cell DNA total amount 0.0064ng (referring to fig. 4).Acute lymphatic leukaemia high pass measures Sequence detects MRD, if MRD > 10-4It (0.01%) is then the positive, if MRD < 10-4It (0.01%) is then feminine gender.Huppert's disease is high Flux sequencing detection MRD, if MRD > 10-5It (0.001%) is then the positive, if MRD < 10-5It (0.001%) is then feminine gender.The present invention Method in use the Hiseq system from illumina company.
Bioinformatic analysis is carried out to lower machine data.Fig. 5 is that treated as a result, result includes for bioinformatics clone count、clone fraction、V segment、D segment、J segment、CDR3 clone sequence、 The information such as CDR3 AA sequence.Fig. 6 is after bioinformatics and visualization processing as a result, wherein each circle represents One clone, the size of each circle indicate the frequency size of the clone, and frequency is bigger, and circle is bigger.
Embodiment 4:B cell acute lymphoid leukemia minimal residual disease MRD detection
B cell acute lymphatic leukaemia minimal residual disease MRD detecting step is divided into two parts, and first part is cancer before treating Cell clonal detection, second part be treatment during or alleviate during minimal residual disease trace detection.
Before treatment, extracted from the fresh bone marrow of B cell acute lymphatic leukaemia patient it is all have core living cells g DNA work It for template, is detected using the kit, specific steps are as shown in previous embodiment 1-3.
Sequencing inspection is carried out to the area IGH, IGH-DJ, IGK, IGL, BCL1/IGH, BCL2/IGH molecule CDR3 in patients B cells It surveys, it is found that (5% judges whether it is cancer cell gram for immune group high-flux sequence to 2 kinds of IGH molecular cloning frequency (being shown in Table 5) > 5% Grand threshold value).As a result it summarizes, identifies conspicuousness and clone and show that high-frequency preceding 10 kinds of clones sort (see Fig. 7).
Cancer cell cloned sequence before table 5 is treated
After treatment, DNA is extracted from the fresh bone marrow of B cell acute lymphatic leukaemia patient as template, using described Kit is detected, and specific steps are as shown in previous embodiment 1-3.
By the 2 kinds of high-frequency cancer cell clones (table 5) detected before tracking treatment, in sample on December 15th, 2017 In detect the minimal residuals of both cancer cells, and both cancer cell clonal levels are below 5%, compare 2017 11 The moon 10, both cancer cells of sample clone frequency was increased, and cancer cell is micro- during depicting treatment according to the result before treatment Small remaining clonal evolution (being detailed in Fig. 8).As the result is shown to detect that residual cancer cell is cloned, cancer cell occupies nucleated cell content For 1.42x10-5, as MRD value.
It should be understood that those skilled in the art can make the present invention various after having read above content of the invention Change or modification, these equivalent forms also fall within the scope of the appended claims of the present application.
SEQUENCE LISTING
<110>Hangzhou Ai Muen Biotechnology Co., Ltd
<120>a kind of kit for detecting minimal residual disease MRD
<130> 18
<160> 253
<170> PatentIn version 3.3
<210> 1
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 1
tcgtgggctc ggagatgtgt ataagagaca gacagcctac atggagctga g 51
<210> 2
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 2
tcgtgggctc ggagatgtgt ataagagaca gacagacctg agcagcctga ca 52
<210> 3
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 3
tcgtgggctc ggagatgtgt ataagagaca gagcagcctg agatctgagg ac 52
<210> 4
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 4
tcgtgggctc ggagatgtgt ataagagaca gacatggacc ctgtggacac a 51
<210> 5
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 5
tcgtgggctc ggagatgtgt ataagagaca gctgtatctg caaatgaaca g 51
<210> 6
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 6
tcgtgggctc ggagatgtgt ataagagaca ggcagattca ccatctccag a 51
<210> 7
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 7
tcgtgggctc ggagatgtgt ataagagaca gagcctgaaa accgaggaca c 51
<210> 8
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 8
tcgtgggctc ggagatgtgt ataagagaca gggccgattc accatctcca ga 52
<210> 9
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 9
tcgtgggctc ggagatgtgt ataagagaca gatcgcctat ctgcaaatga ac 52
<210> 10
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 10
tcgtgggctc ggagatgtgt ataagagaca gagcaaattc accatctcca a 51
<210> 11
<211> 50
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 11
tcgtgggctc ggagatgtgt ataagagaca gagagacaat tccaggaact 50
<210> 12
<211> 49
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 12
tcgtgggctc ggagatgtgt ataagagaca gtgtatctgc aaacgaata 49
<210> 13
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 13
tcgtgggctc ggagatgtgt ataagagaca gctgaagctg agctctgtga c 51
<210> 14
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 14
tcgtgggctc ggagatgtgt ataagagaca gtacctgcag tggagcagcc t 51
<210> 15
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 15
tcgtgggctc ggagatgtgt ataagagaca gtgaactctg tgactcccga g 51
<210> 16
<211> 49
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 16
tcgtgggctc ggagatgtgt ataagagaca gagcctaaag gctgaggac 49
<210> 17
<211> 48
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 17
tcgtgggctc ggagatgtgt ataagagaca ggcggaatgt gtgcaggc 48
<210> 18
<211> 50
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 18
tcgtgggctc ggagatgtgt ataagagaca gactgggctc agagtcctct 50
<210> 19
<211> 48
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 19
tcgtgggctc ggagatgtgt ataagagaca gtggccctgg gaatataa 48
<210> 20
<211> 47
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 20
tcgtgggctc ggagatgtgt ataagagaca gatccccagg acgcagc 47
<210> 21
<211> 50
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 21
tcgtgggctc ggagatgtgt ataagagaca gagggggaca ctgtgcatgt 50
<210> 22
<211> 48
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 22
tcgtgggctc ggagatgtgt ataagagaca gtgaccccag caagggaa 48
<210> 23
<211> 48
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 23
tcgtgggctc ggagatgtgt ataagagaca gacaggcccc ctaccagc 48
<210> 24
<211> 58
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (47)..(47)
<223> r is a, or g
<220>
<221> misc_feature
<222> (53)..(53)
<223> r is a, or g
<400> 24
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nnctgargag acrgtgac 58
<210> 25
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 25
tcgtgggctc ggagatgtgt ataagagaca gttcactctc acaatcagca gc 52
<210> 26
<211> 50
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 26
tcgtgggctc ggagatgtgt ataagagaca gtactttcac catcagcagc 50
<210> 27
<211> 47
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 27
tcgtgggctc ggagatgtgt ataagagaca gggatctggg acggatt 47
<210> 28
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 28
tcgtgggctc ggagatgtgt ataagagaca ggatttcaca ctgaaaatca gc 52
<210> 29
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 29
tcgtgggctc ggagatgtgt ataagagaca gactgaaaat cagcagagtg ga 52
<210> 30
<211> 50
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 30
tcgtgggctc ggagatgtgt ataagagaca gtggaggctg aggatgttgg 50
<210> 31
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 31
tcgtgggctc ggagatgtgt ataagagaca gttcactctc accatcagca g 51
<210> 32
<211> 48
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 32
tcgtgggctc ggagatgtgt ataagagaca gtctgggaca gatttcac 48
<210> 33
<211> 50
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 33
tcgtgggctc ggagatgtgt ataagagaca gtaataacat agaatctgag 50
<210> 34
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 34
tcgtgggctc ggagatgtgt ataagagaca gacaattaat cctgtggaag ct 52
<210> 35
<211> 58
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (46)..(46)
<223> w is a, or t
<220>
<221> misc_feature
<222> (52)..(52)
<223> y is c, or t
<400> 35
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nngatwtcca cyttggtc 58
<210> 36
<211> 58
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (51)..(51)
<223> s is c, or g
<400> 36
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nngatctcca scttggtc 58
<210> 37
<211> 58
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 37
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nnaatctcca gtcgtgtc 58
<210> 38
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 38
tcgtgggctc ggagatgtgt ataagagaca ggaggatgag gctgattatt ac 52
<210> 39
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 39
tcgtgggctc ggagatgtgt ataagagaca gatcactggc ctctggcctg a 51
<210> 40
<211> 49
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 40
tcgtgggctc ggagatgtgt ataagagaca gatcaccgga ctccagact 49
<210> 41
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 41
tcgtgggctc ggagatgtgt ataagagaca gaccaattct ctggttccaa g 51
<210> 42
<211> 49
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 42
tcgtgggctc ggagatgtgt ataagagaca gctccaggct gaggacgag 49
<210> 43
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 43
tcgtgggctc ggagatgtgt ataagagaca gatccctgag cgattctctg g 51
<210> 44
<211> 49
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 44
tcgtgggctc ggagatgtgt ataagagaca gggatgaggc tgactatta 49
<210> 45
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 45
tcgtgggctc ggagatgtgt ataagagaca ggaggatgag gctgattatt ac 52
<210> 46
<211> 49
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 46
tcgtgggctc ggagatgtgt ataagagaca gttctctggc tccagctca 49
<210> 47
<211> 48
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 47
tcgtgggctc ggagatgtgt ataagagaca gatcagcagg gtcctgac 48
<210> 48
<211> 49
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 48
tcgtgggctc ggagatgtgt ataagagaca gggatgaggc tgactatta 49
<210> 49
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (37)..(37)
<223> y is c, or t
<220>
<221> misc_feature
<222> (42)..(42)
<223> r is a, or g
<400> 49
tcgtgggctc ggagatgtgt ataagagaca gtccagyctg argatgaggc t 51
<210> 50
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 50
tcgtgggctc ggagatgtgt ataagagaca gatctctgga ctgaagactg a 51
<210> 51
<211> 48
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 51
tcgtgggctc ggagatgtgt ataagagaca gggcaaagct gccctgac 48
<210> 52
<211> 50
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 52
tcgtgggctc ggagatgtgt ataagagaca gatgatgaat ctgattatta 50
<210> 53
<211> 50
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 53
tcgtgggctc ggagatgtgt ataagagaca gaccatcaag aacatccagg 50
<210> 54
<211> 50
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 54
tcgtgggctc ggagatgtgt ataagagaca gctccagcct gaggacgagg 50
<210> 55
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (51)..(51)
<223> s is c, or g
<400> 55
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nnaggacggt saccttggt 59
<210> 56
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (56)..(56)
<223> b is t, c, or g
<400> 56
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nnaggacggt cagctbggt 59
<210> 57
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 57
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nnaaaatgat cagctgggt 59
<210> 58
<211> 53
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 58
tcgtgggctc ggagatgtgt ataagagaca gagatccggt ccacaaagct gga 53
<210> 59
<211> 55
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 59
tcgtgggctc ggagatgtgt ataagagaca gatcaattcc ctggagcttg gtgac 55
<210> 60
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 60
tcgtgggctc ggagatgtgt ataagagaca gagtcgcttc tcacctgaat gc 52
<210> 61
<211> 50
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 61
tcgtgggctc ggagatgtgt ataagagaca ggctctgaga tgaatgtgag 50
<210> 62
<211> 53
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 62
tcgtgggctc ggagatgtgt ataagagaca gtctgagctg aatgtgaacg cct 53
<210> 63
<211> 49
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 63
tcgtgggctc ggagatgtgt ataagagaca gggctacaat gtctccaga 49
<210> 64
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 64
tcgtgggctc ggagatgtgt ataagagaca ggctgctccc tcccagacat ct 52
<210> 65
<211> 50
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (38)..(38)
<223> r is a, or g
<400> 65
tcgtgggctc ggagatgtgt ataagagaca gggatccrtc tccactctga 50
<210> 66
<211> 49
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 66
tcgtgggctc ggagatgtgt ataagagaca gcggttctct gcagagagg 49
<210> 67
<211> 49
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 67
tcgtgggctc ggagatgtgt ataagagaca ggtctctact ctgaagatc 49
<210> 68
<211> 55
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 68
tcgtgggctc ggagatgtgt ataagagaca ggacttgcac tctgaactaa acctg 55
<210> 69
<211> 50
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 69
tcgtgggctc ggagatgtgt ataagagaca ggctcccaga catctgtgta 50
<210> 70
<211> 53
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 70
tcgtgggctc ggagatgtgt ataagagaca gctcactctg gagtctgctg cct 53
<210> 71
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 71
tcgtgggctc ggagatgtgt ataagagaca gagtagactc cactctcaag at 52
<210> 72
<211> 49
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 72
tcgtgggctc ggagatgtgt ataagagaca gcagggactc agctgtgta 49
<210> 73
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 73
tcgtgggctc ggagatgtgt ataagagaca gactatcatt ctgaactgaa ca 52
<210> 74
<211> 53
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 74
tcgtgggctc ggagatgtgt ataagagaca gaactggagg attctggagt tta 53
<210> 75
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 75
tcgtgggctc ggagatgtgt ataagagaca ggacatccgc tcaccaggcc tg 52
<210> 76
<211> 54
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 76
tcgtgggctc ggagatgtgt ataagagaca ggagatccag gctacgaagc ttga 54
<210> 77
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 77
tcgtgggctc ggagatgtgt ataagagaca gaacggaacg tcttccacgc tg 52
<210> 78
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 78
tcgtgggctc ggagatgtgt ataagagaca gagcatcctg aggatccagc ag 52
<210> 79
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 79
tcgtgggctc ggagatgtgt ataagagaca gctcactgtg acatcggccc aa 52
<210> 80
<211> 53
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 80
tcgtgggctc ggagatgtgt ataagagaca gtctgacagt gaccagtgcc cat 53
<210> 81
<211> 53
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 81
tcgtgggctc ggagatgtgt ataagagaca gtgtaccttg gagatccagt cca 53
<210> 82
<211> 50
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 82
tcgtgggctc ggagatgtgt ataagagaca gtgcagcctg gcaatcctgt 50
<210> 83
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 83
tcgtgggctc ggagatgtgt ataagagaca ggatggatac agtgtctctc ga 52
<210> 84
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (41)..(41)
<223> r is a, or g
<220>
<221> misc_feature
<222> (49)..(49)
<223> y is c, or t
<400> 84
tcgtgggctc ggagatgtgt ataagagaca gctgaccctg ragtctgcya g 51
<210> 85
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 85
tcgtgggctc ggagatgtgt ataagagaca ggaagggtac aaagtctctc ga 52
<210> 86
<211> 53
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 86
tcgtgggctc ggagatgtgt ataagagaca gctgattctg gagtccgcca gca 53
<210> 87
<211> 53
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 87
tcgtgggctc ggagatgtgt ataagagaca gtcaactctg actgtgagca aca 53
<210> 88
<211> 50
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 88
tcgtgggctc ggagatgtgt ataagagaca gcaggaccgg cagttcatcc 50
<210> 89
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 89
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nntacaactg tgagtctgg 59
<210> 90
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 90
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nntacaacgg ttaacctgg 59
<210> 91
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 91
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nntacaacag tgagccaac 59
<210> 92
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 92
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nntacaagac agagagctg 59
<210> 93
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (30)..(33)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (35)..(38)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (40)..(41)
<223> n is a, c, g, or t
<400> 93
tcggcagcgt cagatgtgta taagagacan nnntnnnntn ntacaggatg gagagtcga 59
<210> 94
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (30)..(33)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (35)..(38)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (40)..(41)
<223> n is a, c, g, or t
<400> 94
tcggcagcgt cagatgtgta taagagacan nnntnnnntn ntacacagtg agcctggtc 59
<210> 95
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 95
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nntacacggt gagccgtgt 59
<210> 96
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (48)..(48)
<223> y is c, or t
<400> 96
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nntacagyac ggtcagcct 59
<210> 97
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 97
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nntacactgt cagccgggt 59
<210> 98
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 98
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nntacactga gagccgggt 59
<210> 99
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 99
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nntacaccag gagccgcgt 59
<210> 100
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (30)..(33)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (35)..(38)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (40)..(41)
<223> n is a, c, g, or t
<400> 100
tcggcagcgt cagatgtgta taagagacan nnntnnnntn ntacaccgtg agcctggtg 59
<210> 101
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 101
tcgtgggctc ggagatgtgt ataagagaca gactccagga aagtatgaca ct 52
<210> 102
<211> 49
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 102
tcgtgggctc ggagatgtgt ataagagaca gaaatctaat tgaaaatga 49
<210> 103
<211> 48
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 103
tcgtgggctc ggagatgtgt ataagagaca gttggaatca ggaatcag 48
<210> 104
<211> 49
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 104
tcgtgggctc ggagatgtgt ataagagaca ggaaacgtct acatccact 49
<210> 105
<211> 53
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 105
tcgtgggctc ggagatgtgt ataagagaca gacttcaatc cttaccatca agt 53
<210> 106
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 106
tcgtgggctc ggagatgtgt ataagagaca gttcttagag aaagaagatg ag 52
<210> 107
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 107
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nntgttgttc cactgccaa 59
<210> 108
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 108
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nntgttccgg gaccaaata 59
<210> 109
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 109
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nntgttacta tgagcctag 59
<210> 110
<211> 53
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 110
tcgtgggctc ggagatgtgt ataagagaca gaatccgtcg ccttaaccat ttc 53
<210> 111
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 111
tcgtgggctc ggagatgtgt ataagagaca ggatattgca aagaacctgg ct 52
<210> 112
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 112
tcgtgggctc ggagatgtgt ataagagaca gatctctcca gtaaggactg aa 52
<210> 113
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 113
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nntccacagt cacacgggt 59
<210> 114
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 114
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nntccacgaa gagtttgat 59
<210> 115
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 115
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nntccacgaa gagtttgat 59
<210> 116
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 116
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nntccagata ggttccttt 59
<210> 117
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 117
tctcgtgggc tcggagatgt gtataagaga cagataaagg cgaggagcat a 51
<210> 118
<211> 61
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (30)..(33)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (35)..(38)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (40)..(41)
<223> n is a, c, g, or t
<400> 118
tcggcagcgt cagatgtgta taagagacan nnntnnnntn nttacctgag gagacggtga 60
c 61
<210> 119
<211> 54
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 119
tctcgtgggc tcggagatgt gtataagaga cagaccagca gattcaaatc tatg 54
<210> 120
<211> 54
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 120
tctcgtgggc tcggagatgt gtataagaga cagactctgt ggcattattg catt 54
<210> 121
<211> 54
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 121
tctcgtgggc tcggagatgt gtataagaga cagcacctgc tggatacaac actg 54
<210> 122
<211> 53
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 122
tctcgtgggc tcggagatgt gtataagaga cagtgacaga gcaaaacatg aac 53
<210> 123
<211> 54
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 123
tctcgtgggc tcggagatgt gtataagaga cagtaatgac tggggagcaa atct 54
<210> 124
<211> 54
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 124
tctcgtgggc tcggagatgt gtataagaga cagactggtt ggcgtggttt agag 54
<210> 125
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 125
tctcgtgggc tcggagatgt gtataagaga cagttctgaa agaaacgaaa gc 52
<210> 126
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 126
tctcgtgggc tcggagatgt gtataagaga caggagcaag cgcccaataa at 52
<210> 127
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 127
tctcgtgggc tcggagatgt gtataagaga cagaatgcca tctcaaatcc aa 52
<210> 128
<211> 134
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 128
acagcctaca tggagctgag aagcgtctga gtacatcagt gccgttgcta agtaactact 60
ttggcgcgac agttatctac gtaggtcagg actgcaatag tgaatgtcga cgttgcgtgt 120
caccgtctcc tcag 134
<210> 129
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 129
acagacctga gcagcctgac aaagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgtg 120
tcactgtctc ctcag 135
<210> 130
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 130
agcagcctga gatctgagga caagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgtg 120
tcactgtctc ctcag 135
<210> 131
<211> 134
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 131
acatggaccc tgtggacaca aagcgtctga gtacatcagt gccgttgcta agtaactact 60
ttggcgcgac agttatctac gtaggtcagg actgcaatag tgaatgtcga cgttgcgtgt 120
caccgtctct tcag 134
<210> 132
<211> 134
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 132
ctgtatctgc aaatgaacag aagcgtctga gtacatcagt gccgttgcta agtaactact 60
ttggcgcgac agttatctac gtaggtcagg actgcaatag tgaatgtcga cgttgcgtgt 120
caccgtctcc tcag 134
<210> 133
<211> 134
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 133
gcagattcac catctccaga aagcgtctga gtacatcagt gccgttgcta agtaactact 60
ttggcgcgac agttatctac gtaggtcagg actgcaatag tgaatgtcga cgttgcgtgt 120
caccgtctct tcag 134
<210> 134
<211> 134
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 134
agcctgaaaa ccgaggacac aagcgtctga gtacatcagt gccgttgcta agtaactact 60
ttggcgcgac agttatctac gtaggtcagg actgcaatag tgaatgtcga cgttgcgtgt 120
cactgtctcc tcag 134
<210> 135
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 135
ggccgattca ccatctccag aaagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgtg 120
tcaccgtctc ttcag 135
<210> 136
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 136
atcgcctatc tgcaaatgaa caagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgtg 120
tcaccgtctc ctcag 135
<210> 137
<211> 134
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 137
agcaaattca ccatctccaa aagcgtctga gtacatcagt gccgttgcta agtaactact 60
ttggcgcgac agttatctac gtaggtcagg actgcaatag tgaatgtcga cgttgcgtgt 120
cactgtctcc tcag 134
<210> 138
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 138
agagacaatt ccaggaacta agcgtctgag tacatcagtg ccgttgctaa gtaactactt 60
tggcgcgaca gttatctacg taggtcagga ctgcaatagt gaatgtcgac gttgcgtgtc 120
accgtctctt cag 133
<210> 139
<211> 132
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 139
tgtatctgca aacgaataaa gcgtctgagt acatcagtgc cgttgctaag taactacttt 60
ggcgcgacag ttatctacgt aggtcaggac tgcaatagtg aatgtcgacg ttgcgtgtca 120
ccgtctcctc ag 132
<210> 140
<211> 134
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 140
ctgaagctga gctctgtgac aagcgtctga gtacatcagt gccgttgcta agtaactact 60
ttggcgcgac agttatctac gtaggtcagg actgcaatag tgaatgtcga cgttgcgtgt 120
cactgtctcc tcag 134
<210> 141
<211> 134
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 141
tacctgcagt ggagcagcct aagcgtctga gtacatcagt gccgttgcta agtaactact 60
ttggcgcgac agttatctac gtaggtcagg actgcaatag tgaatgtcga cgttgcgtgt 120
caccgtctcc tcag 134
<210> 142
<211> 134
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 142
tgaactctgt gactcccgag aagcgtctga gtacatcagt gccgttgcta agtaactact 60
ttggcgcgac agttatctac gtaggtcagg actgcaatag tgaatgtcga cgttgcgtgt 120
caccgtctct tcag 134
<210> 143
<211> 132
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 143
agcctaaagg ctgaggacaa gcgtctgagt acatcagtgc cgttgctaag taactacttt 60
ggcgcgacag ttatctacgt aggtcaggac tgcaatagtg aatgtcgacg ttgcgtgtca 120
ctgtctcctc ag 132
<210> 144
<211> 131
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 144
gcggaatgtg tgcaggcaag cgtctgagta catcagtgcc gttgctaagt aactactttg 60
gcgcgacagt tatctacgta ggtcaggact gcaatagtga atgtcgacgt tgcgtgtcac 120
cgtctcctca g 131
<210> 145
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 145
actgggctca gagtcctcta agcgtctgag tacatcagtg ccgttgctaa gtaactactt 60
tggcgcgaca gttatctacg taggtcagga ctgcaatagt gaatgtcgac gttgcgtgtc 120
actgtctcct cag 133
<210> 146
<211> 131
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 146
tggccctggg aatataaaag cgtctgagta catcagtgcc gttgctaagt aactactttg 60
gcgcgacagt tatctacgta ggtcaggact gcaatagtga atgtcgacgt tgcgtgtcac 120
tgtctcctca g 131
<210> 147
<211> 130
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 147
atccccagga cgcagcaagc gtctgagtac atcagtgccg ttgctaagta actactttgg 60
cgcgacagtt atctacgtag gtcaggactg caatagtgaa tgtcgacgtt gcgtgtcacc 120
gtctcttcag 130
<210> 148
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 148
agggggacac tgtgcatgta agcgtctgag tacatcagtg ccgttgctaa gtaactactt 60
tggcgcgaca gttatctacg taggtcagga ctgcaatagt gaatgtcgac gttgcgtgtc 120
accgtctcct cag 133
<210> 149
<211> 131
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 149
tgaccccagc aagggaaaag cgtctgagta catcagtgcc gttgctaagt aactactttg 60
gcgcgacagt tatctacgta ggtcaggact gcaatagtga atgtcgacgt tgcgtgtcac 120
cgtctcttca g 131
<210> 150
<211> 131
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 150
acaggccccc taccagcaag cgtctgagta catcagtgcc gttgctaagt aactactttg 60
gcgcgacagt tatctacgta ggtcaggact gcaatagtga atgtcgacgt tgcgtgtcac 120
tgtctcctca g 131
<210> 151
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 151
ttcactctca caatcagcag caagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgtg 120
accaaggtgg aaatc 135
<210> 152
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 152
tactttcacc atcagcagca agcgtctgag tacatcagtg ccgttgctaa gtaactactt 60
tggcgcgaca gttatctacg taggtcagga ctgcaatagt gaatgtcgac gttgcgtgac 120
caagctggag atc 133
<210> 153
<211> 130
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 153
ggatctggga cggattaagc gtctgagtac atcagtgccg ttgctaagta actactttgg 60
cgcgacagtt atctacgtag gtcaggactg caatagtgaa tgtcgacgtt gcgtgaccaa 120
ggtggaaatc 130
<210> 154
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 154
gatttcacac tgaaaatcag caagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgtg 120
accaaagtgg atatc 135
<210> 155
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 155
actgaaaatc agcagagtgg aaagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgtg 120
accaagctgg agatc 135
<210> 156
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 156
tggaggctga ggatgttgga agcgtctgag tacatcagtg ccgttgctaa gtaactactt 60
tggcgcgaca gttatctacg taggtcagga ctgcaatagt gaatgtcgac gttgcgtgac 120
caaagtggat atc 133
<210> 157
<211> 134
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 157
ttcactctca ccatcagcag aagcgtctga gtacatcagt gccgttgcta agtaactact 60
ttggcgcgac agttatctac gtaggtcagg actgcaatag tgaatgtcga cgttgcgtga 120
ccaaggtgga gatc 134
<210> 158
<211> 131
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 158
tctgggacag atttcacaag cgtctgagta catcagtgcc gttgctaagt aactactttg 60
gcgcgacagt tatctacgta ggtcaggact gcaatagtga atgtcgacgt tgcgtgacac 120
gactggagat t 131
<210> 159
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 159
taataacata gaatctgaga agcgtctgag tacatcagtg ccgttgctaa gtaactactt 60
tggcgcgaca gttatctacg taggtcagga ctgcaatagt gaatgtcgac gttgcgtgac 120
caaggtggag atc 133
<210> 160
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 160
acaattaatc ctgtggaagc taagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgtg 120
acacgactgg agatt 135
<210> 161
<211> 136
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 161
gaggatgagg ctgattatta caagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgta 120
ccaaggtcac cgtcct 136
<210> 162
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 162
atcactggcc tctggcctga aagcgtctga gtacatcagt gccgttgcta agtaactact 60
ttggcgcgac agttatctac gtaggtcagg actgcaatag tgaatgtcga cgttgcgtac 120
caagctgacc gtcct 135
<210> 163
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 163
atcaccggac tccagactaa gcgtctgagt acatcagtgc cgttgctaag taactacttt 60
ggcgcgacag ttatctacgt aggtcaggac tgcaatagtg aatgtcgacg ttgcgtaccc 120
agctgatcat ttt 133
<210> 164
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 164
accaattctc tggttccaag aagcgtctga gtacatcagt gccgttgcta agtaactact 60
ttggcgcgac agttatctac gtaggtcagg actgcaatag tgaatgtcga cgttgcgtac 120
caaggtcacc gtcct 135
<210> 165
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 165
ctccaggctg aggacgagaa gcgtctgagt acatcagtgc cgttgctaag taactacttt 60
ggcgcgacag ttatctacgt aggtcaggac tgcaatagtg aatgtcgacg ttgcgtaccg 120
agctgaccgt cct 133
<210> 166
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 166
atccctgagc gattctctgg aagcgtctga gtacatcagt gccgttgcta agtaactact 60
ttggcgcgac agttatctac gtaggtcagg actgcaatag tgaatgtcga cgttgcgtac 120
caagctgacc gtcct 135
<210> 167
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 167
ggatgaggct gactattaaa gcgtctgagt acatcagtgc cgttgctaag taactacttt 60
ggcgcgacag ttatctacgt aggtcaggac tgcaatagtg aatgtcgacg ttgcgtaccc 120
agctgatcat ttt 133
<210> 168
<211> 136
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 168
gaggatgagg ctgattatta caagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgta 120
ccaaggtcac cgtcct 136
<210> 169
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 169
ttctctggct ccagctcaaa gcgtctgagt acatcagtgc cgttgctaag taactacttt 60
ggcgcgacag ttatctacgt aggtcaggac tgcaatagtg aatgtcgacg ttgcgtacca 120
agctgaccgt cct 133
<210> 170
<211> 132
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 170
atcagcaggg tcctgacaag cgtctgagta catcagtgcc gttgctaagt aactactttg 60
gcgcgacagt tatctacgta ggtcaggact gcaatagtga atgtcgacgt tgcgtaccga 120
gctgaccgtc ct 132
<210> 171
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 171
ggatgaggct gactattaaa gcgtctgagt acatcagtgc cgttgctaag taactacttt 60
ggcgcgacag ttatctacgt aggtcaggac tgcaatagtg aatgtcgacg ttgcgtacca 120
aggtcaccgt cct 133
<210> 172
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (6)..(6)
<223> y is c, or t
<220>
<221> misc_feature
<222> (11)..(11)
<223> r is a, or g
<400> 172
tccagyctga rgatgaggct aagcgtctga gtacatcagt gccgttgcta agtaactact 60
ttggcgcgac agttatctac gtaggtcagg actgcaatag tgaatgtcga cgttgcgtac 120
caaggtgacc gtcct 135
<210> 173
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 173
atctctggac tgaagactga aagcgtctga gtacatcagt gccgttgcta agtaactact 60
ttggcgcgac agttatctac gtaggtcagg actgcaatag tgaatgtcga cgttgcgtac 120
ccagctgacc gtcct 135
<210> 174
<211> 132
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 174
ggcaaagctg ccctgacaag cgtctgagta catcagtgcc gttgctaagt aactactttg 60
gcgcgacagt tatctacgta ggtcaggact gcaatagtga atgtcgacgt tgcgtaccaa 120
ggtgaccgtc ct 132
<210> 175
<211> 134
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 175
atgatgaatc tgattattaa agcgtctgag tacatcagtg ccgttgctaa gtaactactt 60
tggcgcgaca gttatctacg taggtcagga ctgcaatagt gaatgtcgac gttgcgtacc 120
cagctgaccg tcct 134
<210> 176
<211> 134
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 176
accatcaaga acatccagga agcgtctgag tacatcagtg ccgttgctaa gtaactactt 60
tggcgcgaca gttatctacg taggtcagga ctgcaatagt gaatgtcgac gttgcgtacc 120
aaggtcaccg tcct 134
<210> 177
<211> 134
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 177
ctccagcctg aggacgagga agcgtctgag tacatcagtg ccgttgctaa gtaactactt 60
tggcgcgaca gttatctacg taggtcagga ctgcaatagt gaatgtcgac gttgcgtacc 120
aagctgaccg tcct 134
<210> 178
<211> 138
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 178
agatccggtc cacaaagctg gaaagcgtct gagtacatca gtgccgttgc taagtaacta 60
ctttggcgcg acagttatct acgtaggtca ggactgcaat agtgaatgtc gacgttgcgt 120
accagactca cagttgta 138
<210> 179
<211> 140
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 179
atcaattccc tggagcttgg tgacaagcgt ctgagtacat cagtgccgtt gctaagtaac 60
tactttggcg cgacagttat ctacgtaggt caggactgca atagtgaatg tcgacgttgc 120
gtaccagact cacagttgta 140
<210> 180
<211> 137
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 180
agtcgcttct cacctgaatg caagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgta 120
ccagactcac agttgta 137
<210> 181
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 181
gctctgagat gaatgtgaga agcgtctgag tacatcagtg ccgttgctaa gtaactactt 60
tggcgcgaca gttatctacg taggtcagga ctgcaatagt gaatgtcgac gttgcgtacc 120
aggttaaccg ttgta 135
<210> 182
<211> 138
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 182
tctgagctga atgtgaacgc ctaagcgtct gagtacatca gtgccgttgc taagtaacta 60
ctttggcgcg acagttatct acgtaggtca ggactgcaat agtgaatgtc gacgttgcgt 120
accagactca cagttgta 138
<210> 183
<211> 134
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 183
ggctacaatg tctccagaaa gcgtctgagt acatcagtgc cgttgctaag taactacttt 60
ggcgcgacag ttatctacgt aggtcaggac tgcaatagtg aatgtcgacg ttgcgtacca 120
ggttaaccgt tgta 134
<210> 184
<211> 137
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 184
gctgctccct cccagacatc taagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgta 120
ccaggttaac cgttgta 137
<210> 185
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> 1
<222> (7)..(7)
<223> r is a, or g
<400> 185
ggatccrtct ccactctgaa agcgtctgag tacatcagtg ccgttgctaa gtaactactt 60
tggcgcgaca gttatctacg taggtcagga ctgcaatagt gaatgtcgac gttgcgtagt 120
tggctcactg ttgta 135
<210> 186
<211> 131
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 186
cggttctctg cagagaggaa gcgtctgagt acatcagtgc cgttgctaag taactacttt 60
ggcgcgacag ttatctacgt aggtcaggac tgcaatagtg aatgtcgacg ttgcgttcga 120
ctctccatcc t 131
<210> 187
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 187
gtctctactc tgaagatcaa gcgtctgagt acatcagtgc cgttgctaag taactacttt 60
ggcgcgacag ttatctacgt aggtcaggac tgcaatagtg aatgtcgacg ttgcgtacac 120
ggctcaccgt gta 133
<210> 188
<211> 139
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 188
gacttgcact ctgaactaaa cctaagcgtc tgagtacatc agtgccgttg ctaagtaact 60
actttggcgc gacagttatc tacgtaggtc aggactgcaa tagtgaatgt cgacgttgcg 120
taccagactc acagttgta 139
<210> 189
<211> 134
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 189
gctcccagac atctgtgtaa agcgtctgag tacatcagtg ccgttgctaa gtaactactt 60
tggcgcgaca gttatctacg taggtcagga ctgcaatagt gaatgtcgac gttgcgtaca 120
cggctcaccg tgta 134
<210> 190
<211> 138
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 190
ctcactctgg agtctgctgc ctaagcgtct gagtacatca gtgccgttgc taagtaacta 60
ctttggcgcg acagttatct acgtaggtca ggactgcaat agtgaatgtc gacgttgcgt 120
accaggttaa ccgttgta 138
<210> 191
<211> 137
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 191
agtagactcc actctcaaga taagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgta 120
gttggctcac tgttgta 137
<210> 192
<211> 131
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 192
cagggactca gctgtgtaaa gcgtctgagt acatcagtgc cgttgctaag taactacttt 60
ggcgcgacag ttatctacgt aggtcaggac tgcaatagtg aatgtcgacg ttgcgttcga 120
ctctccatcc t 131
<210> 193
<211> 137
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 193
actatcattc tgaactgaac aaagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgta 120
ccagactcac agttgta 137
<210> 194
<211> 138
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 194
aactggagga ttctggagtt taaagcgtct gagtacatca gtgccgttgc taagtaacta 60
ctttggcgcg acagttatct acgtaggtca ggactgcaat agtgaatgtc gacgttgcgt 120
agttggctca ctgttgta 138
<210> 195
<211> 134
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (131)..(131)
<223> r is a, or g
<400> 195
gacatccgct caccaggcct gaagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgta 120
ggctgaccgt rctg 134
<210> 196
<211> 139
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 196
gagatccagg ctacgaagct tgaaagcgtc tgagtacatc agtgccgttg ctaagtaact 60
actttggcgc gacagttatc tacgtaggtc aggactgcaa tagtgaatgt cgacgttgcg 120
taccaggtta accgttgta 139
<210> 197
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 197
aacggaacgt cttccacgct gaagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgtg 120
accaggctca ctgtg 135
<210> 198
<211> 137
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 198
agcatcctga ggatccagca gaagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgta 120
gttggctcac tgttgta 137
<210> 199
<211> 134
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 199
ctcactgtga catcggccca aaagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgtt 120
cgactctcca tcct 134
<210> 200
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 200
tctgacagtg accagtgccc ataagcgtct gagtacatca gtgccgttgc taagtaacta 60
ctttggcgcg acagttatct acgtaggtca ggactgcaat agtgaatgtc gacgttgcgt 120
caccaggctc acggt 135
<210> 201
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (132)..(132)
<223> r is a, or g
<400> 201
tgtaccttgg agatccagtc caaagcgtct gagtacatca gtgccgttgc taagtaacta 60
ctttggcgcg acagttatct acgtaggtca ggactgcaat agtgaatgtc gacgttgcgt 120
aggctgaccg trctg 135
<210> 202
<211> 132
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 202
tgcagcctgg caatcctgta agcgtctgag tacatcagtg ccgttgctaa gtaactactt 60
tggcgcgaca gttatctacg taggtcagga ctgcaatagt gaatgtcgac gttgcgtacg 120
cggctcctgg tg 132
<210> 203
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 203
gatggataca gtgtctctcg aaagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgtg 120
accaggctca ctgtg 135
<210> 204
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (10)..(10)
<223> r is a, or g
<220>
<221> misc_feature
<222> (18)..(18)
<223> y is c, or t
<400> 204
ctgaccctgr agtctgcyag aagcgtctga gtacatcagt gccgttgcta agtaactact 60
ttggcgcgac agttatctac gtaggtcagg actgcaatag tgaatgtcga cgttgcgtac 120
ccggctctca gtg 133
<210> 205
<211> 137
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 205
gaagggtaca aagtctctcg aaagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgta 120
cccagctctc tgtcttg 137
<210> 206
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 206
ctgattctgg agtccgccag caaagcgtct gagtacatca gtgccgttgc taagtaacta 60
ctttggcgcg acagttatct acgtaggtca ggactgcaat agtgaatgtc gacgttgcgt 120
acgcggctcc tggtg 135
<210> 207
<211> 138
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 207
tcaactctga ctgtgagcaa caaagcgtct gagtacatca gtgccgttgc taagtaacta 60
ctttggcgcg acagttatct acgtaggtca ggactgcaat agtgaatgtc gacgttgcgt 120
acccagctct ctgtcttg 138
<210> 208
<211> 132
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 208
caggaccggc agttcatcca agcgtctgag tacatcagtg ccgttgctaa gtaactactt 60
tggcgcgaca gttatctacg taggtcagga ctgcaatagt gaatgtcgac gttgcgtcac 120
caggctcacg gt 132
<210> 209
<211> 136
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 209
actccaggaa agtatgacac taagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgtt 120
tggcagtgga acaaca 136
<210> 210
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 210
aaatctaatt gaaaatgaaa gcgtctgagt acatcagtgc cgttgctaag taactacttt 60
ggcgcgacag ttatctacgt aggtcaggac tgcaatagtg aatgtcgacg ttgcgttatt 120
tggtcccgga aca 133
<210> 211
<211> 132
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 211
ttggaatcag gaatcagaag cgtctgagta catcagtgcc gttgctaagt aactactttg 60
gcgcgacagt tatctacgta ggtcaggact gcaatagtga atgtcgacgt tgcgtactag 120
gctcatagta ac 132
<210> 212
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 212
gaaacgtcta catccactaa gcgtctgagt acatcagtgc cgttgctaag taactacttt 60
ggcgcgacag ttatctacgt aggtcaggac tgcaatagtg aatgtcgacg ttgcgtttgg 120
cagtggaaca aca 133
<210> 213
<211> 137
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 213
acttcaatcc ttaccatcaa gtaagcgtct gagtacatca gtgccgttgc taagtaacta 60
ctttggcgcg acagttatct acgtaggtca ggactgcaat agtgaatgtc gacgttgcgt 120
tatttggtcc cggaaca 137
<210> 214
<211> 136
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 214
ttcttagaga aagaagatga gaagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgta 120
ctaggctcat agtaac 136
<210> 215
<211> 137
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 215
aatccgtcgc cttaaccatt tcaagcgtct gagtacatca gtgccgttgc taagtaacta 60
ctttggcgcg acagttatct acgtaggtca ggactgcaat agtgaatgtc gacgttgcgt 120
acccgtgtga ctgtgga 137
<210> 216
<211> 136
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 216
gatattgcaa agaacctggc taagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgta 120
cacaactcat cgtgga 136
<210> 217
<211> 136
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 217
atctctccag taaggactga aaagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgta 120
tcaaactctt cgtgga 136
<210> 218
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 218
accagcagat tcaaatctat gaagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acggtcaccg 120
tctcctcagg taa 133
<210> 219
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 219
actctgtggc attattgcat taagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgtcac 120
cgtctcctca ggtaa 135
<210> 220
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 220
cacctgctgg atacaacact gaagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acggtcaccg 120
tctcctcagg taa 133
<210> 221
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 221
tgacagagca aaacatgaac aagcgtctga gtacatcagt gccgttgcta agtaactact 60
ttggcgcgac agttatctac gtaggtcagg actgcaatag tgaatgtcga cgtgtcaccg 120
tctcctcagg taa 133
<210> 222
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 222
taatgactgg ggagcaaatc taagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgtcac 120
cgtctcctca ggtaa 135
<210> 223
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 223
actggttggc gtggtttaga gaagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgtcac 120
cgtctcctca ggtaa 135
<210> 224
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 224
ttctgaaaga aacgaaagca agcgtctgag tacatcagtg ccgttgctaa gtaactactt 60
tggcgcgaca gttatctacg taggtcagga ctgcaatagt gaatgtcgac gttgtcaccg 120
tctcctcagg taa 133
<210> 225
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 225
gagcaagcgc ccaataaata agcgtctgag tacatcagtg ccgttgctaa gtaactactt 60
tggcgcgaca gttatctacg taggtcagga ctgcaatagt gaatgtcgac gttgtcaccg 120
tctcctcagg taa 133
<210> 226
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 226
aatgccatct caaatccaaa agcgtctgag tacatcagtg ccgttgctaa gtaactactt 60
tggcgcgaca gttatctacg taggtcagga ctgcaatagt gaatgtcgac gttgtcaccg 120
tctcctcagg taa 133
<210> 227
<211> 47
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 227
caagcagaag acggcatacg agattcgcct tatcgtgggc tcggaga 47
<210> 228
<211> 47
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 228
caagcagaag acggcatacg agatctagta cgtcgtgggc tcggaga 47
<210> 229
<211> 47
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 229
caagcagaag acggcatacg agatttctgc cttcgtgggc tcggaga 47
<210> 230
<211> 47
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 230
caagcagaag acggcatacg agatgctcag gatcgtgggc tcggaga 47
<210> 231
<211> 47
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 231
caagcagaag acggcatacg agataggagt cctcgtgggc tcggaga 47
<210> 232
<211> 47
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 232
caagcagaag acggcatacg agatcatgcc tatcgtgggc tcggaga 47
<210> 233
<211> 47
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 233
caagcagaag acggcatacg agatgtagag agtcgtgggc tcggaga 47
<210> 234
<211> 47
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 234
caagcagaag acggcatacg agatcctctc tgtcgtgggc tcggaga 47
<210> 235
<211> 47
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 235
caagcagaag acggcatacg agatagcgta gctcgtgggc tcggaga 47
<210> 236
<211> 47
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 236
caagcagaag acggcatacg agatcagcct cgtcgtgggc tcggaga 47
<210> 237
<211> 47
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 237
caagcagaag acggcatacg agattgcctc tttcgtgggc tcggaga 47
<210> 238
<211> 47
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 238
caagcagaag acggcatacg agattcctct actcgtgggc tcggaga 47
<210> 239
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 239
aatgatacgg cgaccaccga gatctacact agatcgcgtc ggcagcgtca g 51
<210> 240
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 240
aatgatacgg cgaccaccga gatctacacc tctctatgtc ggcagcgtca g 51
<210> 241
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 241
aatgatacgg cgaccaccga gatctacact atcctctgtc ggcagcgtca g 51
<210> 242
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 242
aatgatacgg cgaccaccga gatctacaca gagtagagtc ggcagcgtca g 51
<210> 243
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 243
aatgatacgg cgaccaccga gatctacacg taaggaggtc ggcagcgtca g 51
<210> 244
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 244
aatgatacgg cgaccaccga gatctacaca ctgcatagtc ggcagcgtca g 51
<210> 245
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 245
aatgatacgg cgaccaccga gatctacaca aggagtagtc ggcagcgtca g 51
<210> 246
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 246
aatgatacgg cgaccaccga gatctacacc taagcctgtc ggcagcgtca g 51
<210> 247
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 247
aatgatacgg cgaccaccga gatctacacg cgtaagagtc ggcagcgtca g 51
<210> 248
<211> 31
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 248
tcgtgggctc ggagatgtgt ataagagaca g 31
<210> 249
<211> 31
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 249
gtcggcagcg tcagatgtgt ataagagaca g 31
<210> 250
<211> 54
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 250
tcgtgggctc ggagatgtgt ataagagaca gagtgtgaca tggtgtatct ctgc 54
<210> 251
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 251
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nnctgaggta gtcagtcag 59
<210> 252
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 252
tcgtgggctc ggagatgtgt ataagagaca gctcactcct tttgcagacc a 51
<210> 253
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (30)..(33)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (35)..(38)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (40)..(41)
<223> n is a, c, g, or t
<400> 253
tcggcagcgt cagatgtgta taagagacan nnntnnnntn nctgaggttt ttctagacg 59

Claims (16)

1. a kind of kit for detecting minimal residual disease MRD, which is characterized in that including building library kit and illumina index Kit, wherein it is described build library kit include detect respectively IgH (VDJ), IgH (DJ), IgK, IgL, TRB, TRG, TRD, The PCR primer group of BCL1/IGH, BCL2/IGH, House Keeping gene and internal reference DNA.
2. the kit of detection minimal residual disease MRD according to claim 1, which is characterized in that the wherein detection IgH (VDJ) in PCR primer, upstream primer sequence is as shown in SEQ ID NO:1-16, downstream primer sequence such as SEQ ID NO:24 It is shown, wherein N:A or T or C or G；R:A or G；W:A or T；Y:C or T；S:C or G；B:T or C or G.
3. the kit of detection minimal residual disease MRD according to claim 1, which is characterized in that the wherein detection IgH (DJ) in PCR primer, upstream primer sequence is as shown in SEQ ID NO:17-23, downstream primer sequence such as SEQ ID NO:24 It is shown, wherein N:A or T or C or G；R:A or G；W:A or T；Y:C or T；S:C or G；B:T or C or G.
4. the kit of detection minimal residual disease MRD according to claim 1, which is characterized in that wherein detect IgK's In PCR primer, upstream primer sequence is as shown in SEQ ID NO:25-34, downstream primer sequence such as SEQ ID NO:35-37 institute Show, wherein N:A or T or C or G；R:A or G；W:A or T；Y:C or T；S:C or G；B:T or C or G.
5. the kit of detection minimal residual disease MRD according to claim 1, which is characterized in that wherein detect IgL's In PCR primer, upstream primer sequence is as shown in SEQ ID NO:38-54, downstream primer sequence such as SEQ ID NO:55-57 institute Show, wherein N:A or T or C or G；R:A or G；W:A or T；Y:C or T；S:C or G；B:T or C or G.
6. the kit of detection minimal residual disease MRD according to claim 1, which is characterized in that wherein detect TRB's In PCR primer, upstream primer sequence is as shown in SEQ ID NO:58-88, downstream primer sequence such as SEQ ID NO:89-100 institute Show, wherein N:A or T or C or G；R:A or G；W:A or T；Y:C or T；S:C or G；B:T or C or G.
7. the kit of detection minimal residual disease MRD according to claim 1, which is characterized in that wherein detect TRG's In PCR primer, upstream primer sequence is as shown in SEQ ID NO:101-106, downstream primer sequence such as SEQ ID NO:107-109 It is shown, wherein N:A or T or C or G；R:A or G；W:A or T；Y:C or T；S:C or G；B:T or C or G.
8. the kit of detection minimal residual disease MRD according to claim 1, which is characterized in that wherein detect TRD's In PCR primer, upstream primer sequence is as shown in SEQ ID NO:110-112, downstream primer sequence such as SEQ ID NO:113-116 It is shown, wherein N:A or T or C or G；R:A or G；W:A or T；Y:C or T；S:C or G；B:T or C or G.
9. the kit of detection minimal residual disease MRD according to claim 1, which is characterized in that wherein detect BCL1/ In the PCR primer of IGH, upstream primer sequence is as shown in SEQ ID NO:117, downstream primer sequence such as SEQ ID NO:118 institute Show, wherein N:A or T or C or G；R:A or G；W:A or T；Y:C or T；S:C or G；B:T or C or G.
10. the kit of detection minimal residual disease MRD according to claim 1, which is characterized in that wherein detect BCL2/ In the PCR primer of IGH, upstream primer sequence is as shown in SEQ ID NO:119-127, downstream primer sequence such as SEQ ID NO: Shown in 118, wherein N:A or T or C or G；R:A or G；W:A or T；Y:C or T；S:C or G；B:T or C or G.
11. the kit of detection minimal residual disease MRD according to claim 1, which is characterized in that wherein build library kit In further includePremix, Multiplex PCR Mix (2X), Nuclease-Free Water, Elution Buffer。
12. the kit of detection minimal residual disease MRD according to claim 1, which is characterized in that the internal reference DNA Sequence is shown in SEQ ID NO:128-226.
13. the kit of detection minimal residual disease MRD according to claim 1, which is characterized in that the House Keeping gene β-actin PCR primer sequence is SEQ ID NO:250-251, the House Keeping gene GAPDH Gene PCR primer sequence is SEQ ID NO:252-253.
14. the kit of detection minimal residual disease MRD according to claim 1, which is characterized in that wherein described Include sequencing primer and label in illumina index kit, the sequence of the sequencing primer and label is SEQ ID NO: Shown in 227-247, the sequence of middle and upper reaches sequencing primer and label is downstream sequencing primer shown in SEQ ID NO:227-238 Sequence with label is shown in SEQ ID NO:239-247.
15. it is according to claim 1 detection minimal residual disease MRD kit, which is characterized in that in kit it is each at The content divided is different according to the different size of kit.
16. the method for a kind of couple of T, B-cell receptor progress high-flux sequence, which is characterized in that any using claim 1-14 The kit.
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Cited By (2)

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Publication number Priority date Publication date Assignee Title
CN114480659A (en) * 2022-04-01 2022-05-13 迈杰转化医学研究(苏州)有限公司 Method for determining minimal residual lesion level based on multiplex amplification sequencing
CN114657254A (en) * 2022-05-17 2022-06-24 至本医疗科技（上海）有限公司 Kit and device for BCR/TCR gene rearrangement detection

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DONGEN JV等: "Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombination in suspect lymphoproliferations:Report of the BIOMED-2 Concerted Action BMH4-CT98-3936", 《LEUKEMIA》 *
LU C等: "The value of detecting immunoglobulin gene rearrangements in the diagnosis of b-cell lymphoma", 《ONCOTARGET》 *
RAESS PW等: "The role of molecular pathology in the diagnosis of cutaneous lymphomas", 《PATHOLOG RES INT》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114480659A (en) * 2022-04-01 2022-05-13 迈杰转化医学研究(苏州)有限公司 Method for determining minimal residual lesion level based on multiplex amplification sequencing
CN114480659B (en) * 2022-04-01 2022-07-12 迈杰转化医学研究(苏州)有限公司 Method for determining minimal residual lesion level based on multiplex amplification sequencing
CN114657254A (en) * 2022-05-17 2022-06-24 至本医疗科技（上海）有限公司 Kit and device for BCR/TCR gene rearrangement detection

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