WO2024049276A1 - Composition pour amplification sélective d'adn cible multiple, et procédé d'amplification l'utilisant - Google Patents

Composition pour amplification sélective d'adn cible multiple, et procédé d'amplification l'utilisant Download PDF

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WO2024049276A1
WO2024049276A1 PCT/KR2023/013102 KR2023013102W WO2024049276A1 WO 2024049276 A1 WO2024049276 A1 WO 2024049276A1 KR 2023013102 W KR2023013102 W KR 2023013102W WO 2024049276 A1 WO2024049276 A1 WO 2024049276A1
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mlt
pcr
target dna
primers
adapter
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정상균
양승경
조선화
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주식회사 키오믹스
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
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    • C40—COMBINATORIAL TECHNOLOGY
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    • C40B40/00—Libraries per se, e.g. arrays, mixtures
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    • C12Q2537/00—Reactions characterised by the reaction format or use of a specific feature
    • C12Q2537/10—Reactions characterised by the reaction format or use of a specific feature the purpose or use of
    • C12Q2537/143—Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
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    • C12Q2549/00—Reactions characterised by the features used to influence the efficiency or specificity
    • C12Q2549/10—Reactions characterised by the features used to influence the efficiency or specificity the purpose being that of reducing false positive or false negative signals
    • C12Q2549/119—Reactions characterised by the features used to influence the efficiency or specificity the purpose being that of reducing false positive or false negative signals using nested primers

Definitions

  • the present invention relates to a composition for selective amplification of multiple target DNA and an amplification method using the same. Specifically, a composition for suppression PCR for effectively amplifying multiple desired targets in a complex DNA sample such as a genome and a composition using the same. It's about method.
  • nucleic acids When nucleic acids are extracted from biological samples such as blood to analyze nucleic acids (DNA or RNA) in living organisms, the amount extracted is generally too small to be directly used for various analyses. Therefore, in order to accurately analyze the extracted nucleic acids, amplification of the extracted nucleic acids is required. need.
  • PCR Polymerase Chain Reaction
  • a representative method of amplifying DNA is a highly efficient amplification technology that selectively amplifies target genes in large quantities, so it is widely used in various fields. It is widely used in.
  • detecting gene mutations related to human cancer development requires high sensitivity and specificity. Since the mutation to be detected belongs to a somatic mutation, it exists in very small amounts mixed with wild-type normal DNA, and is a point mutation or base sequence mutation that does not have a large base sequence difference from the normal gene. This is because false-positives may occur if the target mutation is not accurately recognized in complex insertion/deletion mutations, gene fusion mutations, etc.
  • the important requirements for a tumor-specific mutation detection test are (1) sensitivity to detect mutant DNA that exists at a low rate in normal DNA and (2) incorrectly determining normal DNA as mutant DNA.
  • the goal is to have specificity that can reduce the false-positive rate as much as possible.
  • a variety of testing methods to detect tumor-specific mutations have been developed and used, and representative methods include direct sequencing, allele-specific amplification (AS-PCR), and restriction enzyme fragment length polymorphism. (Restriction Fragment Length Polymorphism; RFLP), TaqMan probe method, ARMS (amplification refractory mutation system) PCR, etc.
  • AS-PCR allele-specific amplification
  • RFLP restriction enzyme fragment length polymorphism
  • RFLP Restriction Fragment Length Polymorphism
  • TaqMan probe method amplification refractory mutation system
  • ARMS amplification refractory mutation system
  • the ARMS-PCR method was developed as a way to improve specificity, which is a drawback of AS-PCR. Like AS-PCR, it is based on the principle that PCR is performed only when the primer consists of a perfectly complementary sequence to the template sequence. (Newton, C. R., et al., 1989. Nucl Acids Res, 17; 2503-2516), has the disadvantage of having to go through experimental trial and error to determine the optimal primer with excellent specificity (Drenkard, E., et al ., 2000. Plant Physiol. 124: 1483-1492).
  • the present inventors have made diligent efforts to develop a method that can dramatically increase the specificity of target nucleic acid amplification that exists at a very low concentration, and as a result, an adapter of a specific structure and multiple target DNA sequences to be amplified are used. It was confirmed that multiple target DNA sequences can be effectively, selectively and specifically amplified when specific primers are included and, in particular, the total amount of primers specific to the adapter is set to a specific range, and the present invention was completed.
  • One object of the present invention is to provide a top oligo and a bottom oligo containing a molecular marker region containing 2 or more N, a sequence region capable of complementary binding to the bottom oligo, and a strand identifier.
  • Adapter containing; PCR primers consisting of sequences specific to part of the adapter sequence; Primers for nested PCR consisting of sequences specific to part of the adapter sequence; and a primer that binds complementary to the target DNA sequence.
  • a composition for selective amplification of multiple target DNA is provided.
  • Another object of the present invention is to form a top oligo and bottom containing a molecular marker region containing two or more N, a sequence region capable of complementary binding to the bottom oligo, and a strand identifier.
  • the present invention provides a kit for selective amplification of multiple target DNA.
  • Another object of the present invention is to form a top oligo and bottom containing a molecular marker region containing two or more N, a sequence region capable of complementary binding to the bottom oligo, and a strand identifier.
  • the purpose of the present invention is to provide the use of a composition for selective amplification of multiple target DNA.
  • Another object of the present invention is to form a top oligo and bottom containing a molecular marker region containing two or more N, a sequence region capable of complementary binding to the bottom oligo, and a strand identifier.
  • the present invention provides a use for library production of a composition comprising a.
  • Another object of the present invention is to manufacture an adapter; Binding the adapter to multiple isolated target DNA sequences; Preparing primers specific for the isolated multiple target DNA sequences and the adapter, respectively; and selectively amplifying the separated multiple target DNA sequences using the prepared primers.
  • Another object of the present invention is to manufacture an adapter; Binding the adapter to multiple isolated target DNA sequences; Preparing primers specific for the isolated multiple target DNA sequences and the adapter, respectively; Obtaining an amplification product by selectively amplifying multiple target DNA sequences separated using the prepared primers; and constructing a library using the obtained amplification products.
  • Another object of the present invention is to provide a library prepared by the above library preparation method.
  • the multi-target DNA amplification method and the target DNA sequencing method effectively amplify the target sequence in samples with high sequence complexity such as genomes or transcripts, and the adapter used in this case is By locating molecular markers and strand identifiers, it is possible to provide a method to precisely and accurately reveal the original sequence structure of the target sequence through high-throughput sequencing and bioinformatics analysis of the amplification product.
  • Figure 1 is a diagram showing the manufacturing process of the adapter manufactured in the example and the configuration and structure of the adapter. Here, the sequence and binding site of the adapter-specific primer are indicated together.
  • Figure 2 is a diagram showing the DNA amplified through primary PCR and nested PCR under each condition after conjugating an adapter to the cut DNA and electrophoresing it on an agarose gel.
  • R100, R200, R300, and R600 represent the experimental group consisting of 100, 200, 300, and 600 primers for target amplification, respectively, and the total amount of primers in the target amplification primer pool applied in each experimental group is expressed as the total amount of primers in each amplified DNA. It is indicated at the top.
  • Figure 3 is a diagram showing the total number of base sequences produced through NGS.
  • Figure 4 is a diagram showing the ratio of base sequences with intact adapter structures.
  • Figure 5 is a diagram showing the ratio of base sequences mapped to the reference genome among base sequences with intact adapter structures.
  • Figure 6 is a diagram showing the mapping pattern of target and non-target regions of the genome mapped base sequence.
  • Figure 7 is a diagram showing the number and ratio of targets with mapped base sequences.
  • Figure 8 is a diagram showing the ratio of base sequences constituting a duplex molecular sequence among base sequences with an intact adapter structure.
  • Figure 9 shows the number and total target sites to which the molecular duplex sequence was mapped for each library when a molecular duplex consensus sequence was constructed by applying a certain standard using the sequences mapped to the reference genome. This is a graph showing the ratio on target according to the minimum number of molecules mapped per target.
  • Figure 10 is a diagram showing the scores calculated for each indicator in highlighted colors in the drawing described above. Specifically, the total range of primers showing excellent performance for each primer pool is indicated with a thick outline.
  • Figure 11 is a diagram showing the amount of individual primers in each mixed group (unit: pmole).
  • Figure 12 is a diagram showing the number of molecular sequences and target capture rate obtained under each condition when duplex molecular sequences were constructed through experiments and analysis performed under given conditions for 1,244 targets.
  • One aspect of the present invention for achieving the above object is a molecular labeling region containing two or more N, a sequence region capable of complementary binding to the bottom oligo, and a top including a strand identifier.
  • Adapter including oligo and bottom_oligo; PCR primers consisting of sequences specific to part of the adapter sequence; Primers for nested PCR consisting of sequences specific to part of the adapter sequence; It provides a composition for selective amplification of multiple target DNA, including a primer that binds complementary to the target DNA sequence.
  • composition for selective amplification of multiple target DNA refers to a use that can specifically amplify a target containing one or more, specifically two or more, known DNA sequences. It refers to a composition with a specific sequence, and is not intended to amplify target DNA limited to a specific sequence. Any sequence containing an isolated known DNA sequence for the purpose of amplification may be included without limitation, and two or more known sequences may be included. Included sequences may also be included without limitation.
  • the term "adapter” refers to a primer that binds to both ends of an isolated DNA fragment (sequence) and uses a primer having a sequence specific to the adapter alone and/or simultaneously with a target sequence-specific primer, thereby forming a gap between each adapter. It may refer to a base sequence containing a double helix structure used to amplify a separated target DNA sequence or to amplify a DNA sequence between an adapter and a separate target DNA sequence-specific primer.
  • the adapter may have a partially single-stranded portion or may be partially double-stranded and composed of non-complementary base pairs.
  • the adapter has a specific structure comprising a top oligo and a bottom oligo containing a molecular marker region containing 2 or more N, a sequence region capable of complementary binding to the bottom oligo, and a strand identifier. You can have
  • top_oligo refers to an oligonucleotide located above an arbitrary phase in an adapter
  • bottom_oligo refers to an oligonucleotide located below an arbitrary phase in an adapter
  • bottom_oligo is a part of the top_oligo. It may include a sequence region that binds complementary to the sequence.
  • the bottom oligo may bind complementary to some sequences of the top oligo, and some may be 5 to 10 or more bases, including 11 or more, 12 or more, 13 or more, 14 or more, 15 or more bases.
  • the length can be included without limitation as long as it is an oligonucleotide that can bind complementary to a part of the sequence of the top oligo, such as 16 or more, 17 or more, 18 or more, 19 or more, or 20 or more oligonucleotides.
  • the top oligo includes at least one, specifically, a molecular marker region containing at least 2 N, a sequence region that can bind complementary to the bottom oligo, and one when complementary to the bottom oligo. More specifically, it may include a strand identifier (strand identifier) that distinguishes DNA strands including one, two, or three mismatches.
  • a strand identifier strand identifier
  • N which constitutes the molecular marker region, causes bases A, G, T, or C to randomly appear at the position occupied by N in the sequence to form various sequence combinations with several bases represented by N, and to the adapter.
  • the purpose is to provide a means to distinguish bound DNA fragments.
  • the adapter may be composed of a top oligo and a bottom oligo, but is not limited thereto.
  • the adapter is composed of only two oligonucleotides, Top_oligo and Bot_oligo, complementary to each other, or is composed of a single strand after Top_oligo and Bottom_oligo are complementary to each other.
  • Top_oligo and Bot_oligo complementary to each other
  • the top and bottom representing the oligo indicate the top and bottom when the double helix DNA, which is a pair of complementary base sequences, is drawn in a drawing, indicating the top and bottom to make it easy to distinguish between the two strands, and indicate the actual top and bottom. It is not pointing.
  • the term "primer” refers to a short-length single-stranded nucleic acid prepared to initiate DNA replication, and is a primer for primary PCR consisting of a sequence specific to a portion of the adapter sequence for the purpose of selective amplification of the multiple target DNA. , adapter-specific nested PCR primers consisting of sequences specific to a portion of the adapter sequence, and primers that bind complementary to the target DNA sequence.
  • primers for PCR consisting of sequences specific to a portion of the adapter sequence and “primers for adapter-specific nested PCR” refer to primers prepared for general PCR and nested PCR, respectively, and bind to the adapter sequence described above. can do.
  • PCR is an abbreviation for polymerase chain reaction and can be used interchangeably with polymerase chain reaction. Specifically, it is a method that can amplify the DNA portion sandwiched between two primers in large quantities in a test tube. 1 Denaturation of the single strand of DNA ⁇ 2 Binding of primers ⁇ 3 Synthesis of complementary DNA by polymerase ⁇ 1 Denaturation ⁇ 2 It refers to a method of repeating the circuit called primer binding....
  • Nested PCR refers to Nested polymerase chain reaction (Nested PCR) and is included in the modification of PCR. Specifically, it is a method to reduce amplification of non-specific binding where primers are attached to base sequences other than the target.
  • Existing PCR requires a complementary primer at the end of the target DNA base sequence, and PCR products are amplified according to the cycle.
  • a common problem is that the primer is attached to an incorrect base sequence site rather than the target template, creating an unwanted strand. appears as the number of PCR cycles increases and was designed to reduce this.
  • the primer for PCR and the primer for nested PCR may be characterized in that they bind to a sequence opposite the junction site with the separated target DNA sequence.
  • the total content of primers for PCR and nested PCR primers may be 25 to 300 pmole under 20ul PCR conditions, and when the amount of the PCR reaction solution is changed, the total content of primers under the 20ul condition is proportional to the amount of the reaction solution. The content may be adjusted in the same way.
  • the primers for PCR and the primers for nested PCR may be characterized as having an individual content of 0.167 to 1.0 pmole under the condition of 20ul of the PCR reaction solution, and the individual content is also proportional to the amount of the reaction solution when the amount of the PCR reaction solution is changed. Therefore, the content may be adjusted to be the same as the individual content in the 20ul condition.
  • the “primer that specifically binds to the target DNA sequence” is according to the primer definition described above and may be a short-stranded primer that can bind complementary to the isolated target DNA sequence to be amplified.
  • the prepared primers may be characterized in that they form a pool of a forward group and a reverse group, respectively, to prevent amplification by the forward primer and reverse primer.
  • the primer pool that specifically binds to the target DNA sequence may include 10 to 10,000 primers, specifically 15 to 8,000, 20 to 6,000, 30 to 4,000, and 100 to 3,000. The number may vary depending on the target DNA sequence, and the forward and reverse primer sequences that specifically bind to the target DNA sequence may be variously composed of some sequences specific to the target DNA sequence.
  • multiple target DNA sequence may refer to an isolated DNA sequence to be selectively amplified.
  • the target DNA sequence may be isolated DNA extracted from body fluid, and the body fluid may be cells, tissues, or blood, but is not limited thereto.
  • the target DNA extracted from the body fluid may be isolated DNA in an extracted state or isolated DNA that has undergone additional cleavage through a physical or chemical method.
  • the extracted target DNA itself or the additionally cut DNA can be subjected to an additional step of cleaning up the separated cleaved ends.
  • the terminal region is cleaned up by A-tailing to achieve suppression. It may be suitable for PCR, but is not limited to this.
  • multiple target DNA sequences may be included without limitation as long as they are known isolated sequences, such as synthetic or manufactured sequences or sequences that have already been manufactured and sold.
  • an adapter Binding the adapter to multiple isolated target DNA sequences; Preparing primers specific for the isolated multiple target DNA sequences and the adapter, respectively; and selectively amplifying the separated multiple target DNA sequences using the prepared primers.
  • an adapter Binding the adapter to multiple isolated target DNA sequences; Preparing primers specific for the isolated multiple target DNA sequences and the adapter, respectively; Obtaining an amplification product by selectively amplifying multiple target DNA sequences separated using the prepared primers; and constructing a library using the obtained amplification products.
  • the terms “adaptor,” “isolated multiple target DNA sequence,” and “isolated multiple target DNA sequence and primers specific for the adapter” are as described above.
  • the above step may be provided as a step of preparing a library for Next Generation Sequencing (NGS).
  • NGS Next Generation Sequencing
  • next Generation Sequencing refers to a high-speed analysis method for the base sequence of the genome, which includes high-throughput sequencing and massive parallel sequencing. ) or can be used interchangeably with second generation sequencing.
  • the term “library” refers to a set of DNA fragments that have been cut with restriction enzymes or the like or have common genetic or structural characteristics and are prepared for determining the base sequence through NGS, but are not limited thereto. Specifically, in the present invention, the library can be prepared through the above steps.
  • two top oligos and bottom oligos are each manufactured, and then the temperature of the solution in which the two oligos are dissolved at the same molar ratio is raised to 90°C or more, specifically 95°C, and then slowly cooled. Their complementary combination can be completed. After the two oligos form complementary bonds, the single-stranded region can undergo a fill-in reaction using DNA polymerase enzyme and dNTP as a substrate to form a complete double-stranded structure. At this time, the 3'-end of the top oligo is composed of base T and does not form a complementary binding pair with the bottom oligo, thereby creating a 3'-T overhang at the end of the adapter to bind to the A-tailed DNA.
  • the adapter can be completed ( Figure 1).
  • Multiple target isolated DNA fragments to be bound to the adapter can be collected from samples of various forms or states such as tissues, cells, and body fluids, and depending on the state of the collected DNA, additional cuts can be performed or the collected DNA can be used as is. Additional cleavage can be done using a physical method using ultrasound to cut into a certain size range, or by using an enzyme system consisting of one or more endonuclease cocktails.
  • A-tailing can be performed, and this can be done according to a conventional method.
  • DNA fragments with 3' A-tails at both ends can be linked using the adapter and DNA ligase described above.
  • the amplification step may be to specifically amplify a plurality of target sequences using the principle of PCR suppression by targeting adapter-bound DNA.
  • Adapter-specific primers can be prepared as primary PCR primers and nested PCR primers to bind to the opposite side of the junction with the cut DNA.
  • Target sequence-specific primers can be used to produce primary PCR primers and nested PCR primers, respectively, so that the base sequence region to be captured can be amplified by PCR.
  • Target sequence-specific primers can be prepared by dividing and mixing them into a primary PCR primer pool and a secondary PCR primer pool according to sequential PCR steps.
  • the target sequence-specific primers By dividing the target sequence-specific primers into a forward group and a reverse group according to the direction of progress to form a pool, it is possible to prevent the forward and reverse primers from existing nearby and amplifying the base sequence due to them.
  • the first PCR can be performed under given conditions using the previously prepared adapter-DNA conjugate as a template, and using adapter primers for the first PCR and primers for the target sequence. Subsequently, the target sequences can be amplified by using the amplification product of the first PCR as a template and performing nested PCR under given conditions using adapter primers for nested PCR and primers for the target sequence.
  • a sequencing library can be constructed using the nested PCR product itself or an amplification product obtained through additional PCR.
  • an NGS library can be constructed by conjugating another adapter to the nested PCR product and then performing additional PCR.
  • amplification product refers to the result of PCR performed using primers, and can be used interchangeably with amplified DNA.
  • the amplified DNA may contain different molecular markers and strand identifiers depending on each template DNA subject to amplification.
  • an NGS process may be additionally performed.
  • oligonucleotides for adapter production were prepared.
  • a site that allows Bot_oligo to bind complementary to Top_oligo to form a partial double helix structure and 2) 8 random oligos.
  • the strand origin of the sequences amplified from target DNA A strand identifier was installed to distinguish origin.
  • top oligo and bottom oligo that make up the adapter were manufactured at the request of Bioneer Co., Ltd. Equal amounts of 50 ⁇ l each of the two oligonucleotides were mixed at a concentration of 100 pmole/ ⁇ l. Then, it was left in sequence at 95°C for 5 minutes and at 75°C for 5 minutes, and then the temperature was lowered from 75°C to 35°C at a rate of 1°C/cycle/min to induce complementary bonding between the two base sequences, resulting in partial duplication. Stranded DNA was produced.
  • Top_Oligo and Bottom_Oligo are as follows:
  • Top_Oligo SEQ ID NO: 2773: 5'-AGGACCGTGTGCTGACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNCTGACGTGACTGACT-3'
  • Bot_Oligo SEQ ID NO: 2774: Phosphate-GTCTGTCACGTCAG-3'.
  • 100ng of genomic DNA of the HCT116 cell line was cut with the restriction enzyme BstNI, and then end polish and 3'-A tailing were performed. End-polish is to make both ends of DNA blunt ends and add phosphate to the 5'-end. 3 units of T4 DNA polymerase are added to 100 ng of cut DNA. 10 units of T4 polynucleotide kinase, 1 ul of 10mM dNTP, 2.5 ul of 10mM ATP, and 2.5 ul of buffer were added and reacted at 25°C for 20 minutes.
  • the adapter-specific primer has the following sequence:
  • Nested PCR primer (SEQ ID NO: 2776): 5'-ACTCTTTCCCTACACGACGCTCTTCCGAT-3'.
  • Target sequence-specific primers were designed with reference to the base sequence of the human reference genome hg38, and through suppression PCR using these target-specific primers, specific exon portions of each gene in 157 randomly selected gene regions were identified by the target primers. It was allowed to be specifically amplified.
  • Target sequence-specific primers (see Table 1 below, SEQ ID NO: 1 to SEQ ID NO: 2772) were divided into groups of 100, 200, 300, 600, and 1,250 inclusion sites, and the primers were divided into primary PCR and nested PCR. After mixing, the PCR reaction was performed by varying the total amount of primers according to the conditions.
  • Target sequence specific primer in the table below, the mlt_1 sequence is set to SEQ ID NO: 1 and the sequence numbers increase by one in the order of the table.
  • mlt_2772 means SEQ ID NO: 2772)
  • Name primer sequence Role of primers in each group R100 R200 R300 R600 R1244 mlt_1 ggcagtcccagcctacCT R R R R mlt_2 TGTCACTGATTTCCTTCACCG R R R R R R mlt_3 gcggtaccgacactgtgg R R R R R R R mlt_4 cgtccccacgtcctgagc R R R R R mlt_5 ccgcagttccagctcccc R R R R R R mlt_6 cagggggatgcacgcaga R R R R mlt_7 GGTCTCTCAACAGGCA
  • R.N. R.N. R.N. R.N. mlt_630 acactgtggccttgtttcct R.N. R.N. R.N. R.N. R.N. mlt_631 cacacgcaatgctgtgtcc R.N. R.N. R.N. R.N. mlt_632 gttccagctccccttccttttg R.N. R.N. R.N. R.N.N.
  • R.N. mlt_638 ccccctaactttggccagag R.N. R.N. R.N. R.N. R.N. mlt_639 accttgccagcatcctcac R.N. R.N. R.N. R.N. R.N. mlt_640 ggagaactctggtttggcca R.N. R.N. R.N. R.N. R.N. mlt_641 CGGGAGCCACTCAGCTACT R.N.N. R.N. R.N. R.N. mlt_642 ctgaatggggagtgcggg R.N. R.N.
  • R.N. R.N. mlt_702 CACCCCCAGCAATGAGAGTA
  • R.N. R.N. R.N. R.N. mlt_703 tgcgggatcatgacttagagc
  • R.N. R.N. R.N. R.N. R.N. mlt_704 GAGCTGCTTCCACGGCTG
  • R.N. R.N. R.N. R.N. mlt_705 ctctggatccccattcttga R.N. R.N. R.N. R.N. mlt_706 ATATTTGGTTCTGCGGCCTGT R.N. R.N.
  • R.N. R.N. mlt_707 GTGACTCCTGAGAAGGTCTGC R.N. R.N. R.N. R.N. mlt_708 atgttagaataacctctgctcca R.N. R.N. R.N. R.N. R.N. mlt_709 ttgagtaagaatcctctctgccta R.N. R.N. R.N. R.N. mlt_710 ttccttgcttccccgctctgctctg R.N. R.N. R.N.
  • R.N. mlt_716 aggttgtttttcagtctttattcaa R.N. R.N. R.N. R.N. R.N. mlt_717 cgaggccagagtcctttagc R.N. R.N. R.N. R.N. R.N. mlt_718 CTCTTCATGGGCTATTCCACA R.N.N. R.N. R.N. R.N. mlt_719 cacttggagtaacaattgcctttt R.N. R.N. R.N. R.N.N.
  • mlt_720 ACTTGGTAGACGGGACTCG R.N. R.N. R.N. R.N. R.N. mlt_721 cccctttttggcccatcct R.N. R.N. R.N. R.N. mlt_722 aacacagcctgcttagtacc R.N. R.N. R.N. R.N. R.N. mlt_723 aagcttctgggttttgcaca R.N. R.N. R.N. R.N. N.
  • mlt_744 caacctcatctgctctttcttgt R.N. R.N. R.N. R.N. mlt_745 ggacttacCATGACTTGCAGC R.N. R.N. R.N. R.N. mlt_746 TGTTTTGCTTTTGTCTGTTTTCC R.N. R.N. R.N. mlt_747 ttcatccattcctgcactaatgt R.N. R.N. R.N. mlt_748 tgtgcaacattttgacatgg R.N. R.N. R.N.
  • R.N. R.N. R.N. mlt_800 cagagccgatggtccgaTC R.N. R.N. R.N. R.N. mlt_801 tttgaacatttctccattttcc R.N. R.N. R.N. mlt_802 tggacggacatttttcctcc R.N. R.N. R.N. mlt_803 gatttgcccaattgttgcttt R.N. R.N. R.N. R.N.
  • mlt_808 ctcaggcagccttgagtcaa R.N. R.N. R.N. R.N. mlt_809 aagacccATGTTTGTATTGCTG R.N. R.N. R.N. R.N. mlt_810 ggagagcattcctcctccatg R.N. R.N. R.N. R.N. mlt_811 tcagtgatcgcctcttgtga R.N. R.N. R.N. mlt_812 TCACAGTGGCAATGGAACCT R.N. R.N. R.N. R.N.
  • mlt_840 cccctccccccgtatctccccccgtatctcccc R.N. R.N. R.N. mlt_841 aaaatgctggctgacctaaagc R.N. R.N. R.N. mlt_842 cagcttggcctcagtacaa R.N. R.N. R.N. mlt_843 acagaccccaccagtcactcac R.N. R.N. R.N. mlt_844 tcccaatggaagcacagactg R.N. R.N. R.N.
  • mlt_845 acagcaaacaaccagacacaca R.N. R.N. R.N. mlt_846 TTTGGTGGCTGCCTTTCTG R.N. R.N. R.N. mlt_847 gggctcatactatcctccgt R.N. R.N. R.N. mlt_848 ATCCCTGTGTTCATTCCCAT R.N. R.N. mlt_849 ccctgtgggtccacttacCT R.N. R.N. R.N. mlt_850 acttacTGGAGTTGCAGGCTG R.N. R.N. R.N.
  • mlt_900 cctttttgttctggcggtgtt R.N. R.N. R.N. mlt_901 CACCCGGGATGGGAGTACTC R.N. R.N. R.N. mlt_902 gctgcccatgagttagagga R.N. R.N. R.N. mlt_903 tctcctgtgtctgtttctagCA R.N. R.N. R.N. mlt_904 taacagacccacgctgtacac R.N. R.N. mlt_905 cagGAGCACCATACTGGACC R.N.
  • mlt_912 AAAATAGCATCCGCCACAAC R.N. R.N. mlt_913 TTGTAGAGGATCTTGAGCGTCTC R.N. R.N. R.N. mlt_914 gctgaatacgccaccttcct R.N. R.N. mlt_915 tgttcatctttcagGGACAGG R.N. R.N. R.N. mlt_916 CTCCGGGCTCCATGAGTG R.N. R.N. R.N. mlt_917 CCGGAGAGGGTACTTTTCCT R.N. R.N.
  • mlt_924 aacgcttcatgctccactact R.N. R.N. mlt_925 gagtggtgctctctctctgcag R.N. R.N. R.N. mlt_926 gtcctgcccttgcacagat R.N. R.N. R.N. mlt_927 cctgcagTCCATCTCAGGTT R.N. R.N. R.N. mlt_928 gactggagctcctcgcta R.N. R.N. R.N.
  • mlt_929 agcccacacatttgcactcta R.N. R.N. R.N. mlt_930 ctcgttcgctctccagcttg R.N. R.N. R.N. mlt_931 cacgaccccattatctgtccc R.N. R.N. mlt_932 ctcacttagtggctgcactca R.N. R.N. mlt_933 tgtcaggagtcagggacgat R.N. R.N. mlt_934 gagaaggagagtgtctgtcgcgc R.N. R.N.
  • mlt_941 aagggtgattccttggcgag R.N. R.N. mlt_942 AATGGTGCAGGCTCCAAGTA R.N. R.N. mlt_943 AATGCAGAAGATTCTTATAAAGTGC R.N. mlt_944 tatgctctgaccccaaacaccc R.N. mlt_945 TTTTGCAGAAGATGAAGTGG R.N. R.N. mlt_946 aagagtacCTGTGCTTTTGGGC R.N. R.N. mlt_947 tccaaagaagctttattggca R.N.
  • mlt_948 CAGTAACCACTGTCCTTTGCAA R.N. mlt_949 GGATGATCTGTTGCTCATTCC R.N. R.N. mlt_950 GGTTCTGTGGGAGATGTTGA R.N. R.N. mlt_951 ATTGAGAAGTTGGCAAGCTTG R.N. R.N. mlt_952 caacatgaaatcccacagacc R.N. R.N. mlt_953 tatttcagGGTGTTGATGATGC R.N. mlt_954 atctcaaacttcttgcacatggc R.N. R.N.
  • mlt_987 acatcccgtccacatatagct R.N. mlt_988 CTTCTGACATTTCAGTAAAGGGC R.N. R.N. mlt_989 AATTTGACGTTTCGAATCTTCAG R.N. mlt_990 gtgcctgaaaagacgacttacC R.N. mlt_991 aacctgccaaacaaagccct R.N. mlt_992 aacatttgttcctcacctgca R.N. R.N. mlt_993 agcacaacaacagtgagctct R.N.
  • mlt_1014 AAGAAATTGAGTTCCGCAGTTGT R.N. R.N. mlt_1015 ACCCACCTCTTGATGAACCA R.N. R.N. mlt_1016 GTGCACTCATTTGTGGACGTCC R.N. R.N. mlt_1017 GAGCCTTTGGTTGCATGCTC R.N. mlt_1018 ATACAGACTTTTGACTGGCGTTG R.N. R.N. mlt_1019 GTGAGGCCGCTTATAACCAA R.N. mlt_1020 aacttacCCTTCAGGAGTCGT R.N. R.N.
  • mlt_1040 caacatggcaaaccggatgg R.N. R.N. mlt_1041 AGCCACAAAACTTACAGATGCAG R.N. R.N. mlt_1042 CCTGAATTTCACTGTGGGCG R.N. R.N. mlt_1043 ATGGCTACCTGTCAGACGTG R.N. R.N. mlt_1044 gggttgcctccagctggt R.N. R.N. mlt_1045 ctccagactgagcacccgt R.N. R.N. mlt_1046 AACCGGCAGTGTGTGCAG R.N. R.N.
  • mlt_1107 agATGTTATGGAAGCAAGTTCACA R.N. mlt_1108 CTGCGGTGAATGACGATG R.N. R.N. mlt_1109 AACTGCACAGTCCATCCTTTG R.N. R.N. mlt_1110 GTAATTTCACATCCAATTTGGGA R.N. R.N. mlt_1111 GTCGTTGTGCTGCACAAGG R.N. R.N. mlt_1112 tgggggaaggtttttcaggg R.N. R.N. mlt_1113 aaacgtgttgtcaagTCATGGA R.N. R.N.
  • mlt_1206 accgtctgattaggagggcct R.N. R.N. mlt_1207 tcttgttccatctgaatggatg R.N. mlt_1208 cctgtgtctgacattccccc R.N. R.N. mlt_1209 attttagGCTGCTGCTCCTGCGT R.N. R.N. mlt_1210 CAGGGCTCCATCCTCTCCA R.N. mlt_1211 ccaaaaagggacggagggtc R.N. R.N.
  • mlt_1238 CACTATGGTAACCAAGCTCCA R.N. mlt_1239 ggcctcttgaagcctctgtt R.N. mlt_1240 ttctgcccttgtctctaagCA R.N. mlt_1241 aggctcttctcaacgggttc R.N. mlt_1242 cagacgtttcctgttgacc R.N. mlt_1243 cagCACCTCTTGGACAGGATT R.N. mlt_1244 ccttcgagcgagagaATGGC R.N.
  • mlt_2020 ctgagaggagcgcgtgag R.N. mlt_2021 ctgagctcctctcctccgta R.N. mlt_2022 cggtttcctctctgtgaag R.N. mlt_2023 atccccgacctgtgt R.N. mlt_2024 tgagatccgttgacttttcca R.N. mlt_2025 ggggcctgacctcctct R.N.
  • mlt_2032 caaagttttaataatttcccctacg R.N. mlt_2033 tccttatttattaattaatgtgcttcttc R.N. mlt_2034 gatgcttaatgccatctcca R.N. mlt_2035 ttacatccctctctgctctgc R.N. mlt_2036 atgtgcttagcccactggaa R.N. mlt_2037 cagtttgctcctcacacacaca R.N.
  • mlt_2045 GCCCACTTGACCACGTGTA R.N. mlt_2046 ggcaaaagtggtcctctctg R.N. mlt_2047 CCGTTCGAGTTCTTCAGGTC R.N. mlt_2048 tggacagcaacaagggtcaa R.N. mlt_2049 GCTGATACGAAGGTTGGGGCA R.N. mlt_2050 caagtcagcctttaagttccttg R.N. mlt_2051 atgacaaggtgaagccaagg R.N.
  • mlt_2060 tttgtatgctgagaagcatgg R.N. mlt_2061 ctctgtgtgacgggaggtgg R.N. mlt_2062 CGGATGTGATAAGGGTGTGTG R.N. mlt_2063 tggttccaactcacCCATAA R.N. mlt_2064 tctcttctgtctcaggggcat R.N. mlt_2065 ccttgaatctgcccctctc R.N. mlt_2066 tgattaaacgccccttacCAC R.N.
  • mlt_2081 GCCCACCTAATTGTACTGAATTG R.N. mlt_2082 GGAACTAACCAAACGGAGCA R.N. mlt_2083 aaaggagagagcagctttcacta R.N. mlt_2084 ttgctgcttactcattgcattt R.N. mlt_2085 tgcctttaacctctgtgctg R.N. mlt_2086 ACCTGACCTTCTTGAAGGAGC R.N. mlt_2087 caggtgtgagcaccacttcc R.N.
  • mlt_2088 ctgccacattgtttccttca R.N. mlt_2089 catgcaaatggtggcttctcc R.N. mlt_2090 atgtgttgggtcgtattgga R.N. mlt_2091 acagccctgcgtgtctc R.N. mlt_2092 ctgagtgggatggtcaaccc R.N. mlt_2093 tgaacaaggtctggctttgc R.N.
  • mlt_2094 agggcttttctcttttcagc R.N. mlt_2095 cactcacatgcatgctttcc R.N. mlt_2096 cccgtaacgtgtgtgtgtgtct R.N. mlt_2097 tctctcccatctcgtctcc R.N. mlt_2098 AAGAAGCTGACGATTCTTCCA R.N. mlt_2099 TCCCGAGATGAAGCTCAGAC R.N. mlt_2100 cactctgaatccccagtgc R.N.
  • mlt_2108 cctcttccctgccttgaact R.N. mlt_2109 gcgaacaggaagactcaagc R.N. mlt_2110 tttacCTCAAGTTGGCTGCA R.N. mlt_2111 aaaagaacgctgagatggatg R.N. mlt_2112 tctttggtggagaaggatgg R.N. mlt_2113 ATCCCGACTTGCTGGAGACT R.N. mlt_2114 ATAATTGCTCGAGGTATGAGATCG R.N.
  • mlt_2122 AGCAGGGAAGGCCAAGG R.N. mlt_2123 GCCGGGAGAACTCTAACTCC R.N. mlt_2124 gaaaatcggtactctttcactcaaga R.N. mlt_2125 tgaaaatcaatgaacacacCTG R.N. mlt_2126 CCCCTGTCCAGTCTCCAC R.N. mlt_2127 ctgtcccctttccttgctt R.N. mlt_2128 ctaaagcccctctctctctg R.N.
  • mlt_2157 cagcctgtaactgaccttgg R.N. mlt_2158 TCTGGAGATGCTGACTTAGTGC R.N. mlt_2159 tctgtctctctgtcctagGGC R.N. mlt_2160 caacgaactggtccctttgt R.N. mlt_2161 ATTACCCAATGGGGACTTGG R.N. mlt_2162 aaacaggctaagcccactga R.N. mlt_2163 ccacctctggcctcacag R.N.
  • mlt_2178 catgttcagaataccctaagtcca R.N. mlt_2179 AGCTGAAGTACTTGGCTGGTC R.N. mlt_2180 catgtcaaagccagaagcag R.N. mlt_2181 aaaagccaaagcgtactgact R.N. mlt_2182 atcgtggccaaacccaaaga R.N. mlt_2183 catacCTCTGGTTGGTGGA R.N. mlt_2184 aaaagccattatcaaaatatgcttac R.N.
  • mlt_2185 ACTGGGACCTCTGGTCATGG R.N. mlt_2186 TTCAAAGGTTGACCATGCTG R.N. mlt_2187 TGTCCCATATTTCCTTGTTGC R.N. mlt_2188 aaaccacaaacggctactgc R.N. mlt_2189 aaggaggtgggctcactaa R.N. mlt_2190 ttgcttagggagcaatggac R.N. mlt_2191 tggattcttttcccttcca R.N. mlt_2192 TGGTCCAGTTCAAAGAGTGG R.N.
  • mlt_2200 agctgccattaaatgctccct R.N.
  • mlt_2201 aagcagacaacaagttgcaga R.N.
  • mlt_2202 aagaagttgtgggtaggatgg R.N.
  • mlt_2203 ttttctcacttcccctcct R.N.
  • mlt_2204 gggtaggacactcaatcttgga R.N. mlt_2205 tggctacatctcttcttgattttc R.N.
  • mlt_2226 tggggattagctgcgtagag R.N. mlt_2227 aggctgatgcttcctcattc R.N. mlt_2228 cagcccatttgtaatgttttatg R.N. mlt_2229 catttgctgaggtggaggat R.N. mlt_2230 agccaacgccttctctcac R.N. mlt_2231 tgaaatgtgaatgacctttttaac R.N.
  • mlt_2301 tgaaggtgcagagctgtttatc R.N.
  • mlt_2302 ccccagctctgttttgagag R.N.
  • mlt_2303 agggcaggaaagagcacata R.N.
  • mlt_2304 ttcctgtgtcgtctagcctttt R.N.
  • mlt_2305 cctccactgacctttgttgg R.N. mlt_2306 gtgcctagtgggtcccttct R.N.
  • mlt_2321 gtgcagagtcccacaggtc R.N. mlt_2322 cagtgccttgcccttttgt R.N. mlt_2323 ttctggccgggacacac R.N. mlt_2324 cacagccttgagccttgc R.N. mlt_2325 cagaaacatgcttcctccaa R.N. mlt_2326 CAAATTTGTAAGCATATTCTTTTGC R.N. mlt_2327 AGTAGCTGCCTAAGTGTGAAGG R.N.
  • mlt_2347 aggtgtgcatgtatcaacattatca R.N. mlt_2348 GCCATGATGCATTGCTCTTA R.N. mlt_2349 CTGCAGTCTGGCTTTCCTCA R.N. mlt_2350 AGAAGGGCTGGCAAAATAGC R.N. mlt_2351 cgccggctttgtcATGATGG R.N. mlt_2352 TCATGACGCTGCCCATCATT R.N. mlt_2353 CCTGTTTTCTGAGACCACGA R.N. mlt_2354 gctacgtgcaaggcattacag R.N.
  • mlt_2355 agcaaataaatatgacaatttcagc R.N. mlt_2356 ttctcatccttcctttggaaaa R.N. mlt_2357 AACACTGGGGCACTTTGTTC R.N. mlt_2358 CGAGCAGCAGCAGCTTGA R.N. mlt_2359 GGTTGAGCTGCAGGATGGT R.N. mlt_2360 tgaaacagccaatgtgtgtc R.N. mlt_2361 ctcattgtgtcttcctctctctctctctctctctct R.N.
  • mlt_2362 CAGAAGCCTCCCAAGCTG R.N. mlt_2363 TGGGACCTGTAAGAGAAGTTGAC R.N. mlt_2364 AGATGGATTCTTGCGTCTGG R.N. mlt_2365 tcttcatgtttgtttggtttgt R.N. mlt_2366 cccgccgttgtacCTATT R.N. mlt_2367 catcatctcctgctgtgg R.N. mlt_2368 AAGAGGCCATCTGGGTGGA R.N.
  • mlt_2396 aagccataacaacagtcttctgtg R.N. mlt_2397 ttttgaggaaatgcatgtgg R.N. mlt_2398 gccataggagattagtcagca R.N. mlt_2399 agaacctttccttcattgtca R.N. mlt_2400 agtcagctttagttgctaaaatcc R.N. mlt_2401 AGTGACCAACATGGAGTCGTG R.N.
  • mlt_2409 acaaacacgagccacaacttt R.N.
  • mlt_2410 acagagcctaaacatcccctta R.N.
  • mlt_2411 TTAATGAGGAGACCCGAAGG
  • mlt_2412 agcatggccccagagtattt R.N.
  • mlt_2413 gcaaggagcatttggggga R.N. mlt_2414 tttcctgctttcaaatgctgt R.N. mlt_2415 ACTCTGATCCTGTGGACTCCA R.N.
  • mlt_2423 CACCTGTCCTTCAGCTGATCT R.N. mlt_2424 AAATCCTGATTGTTGCCATGA R.N. mlt_2425 GAAGTTGGCAAGCTTGGATT R.N. mlt_2426 tcttaaaagacgaattcttcagc R.N. mlt_2427 tcttttagggatgatgagaatgtt R.N. mlt_2428 gactgctccccactcaagac R.N. mlt_2429 atgtggtggagacacagtgg R.N.
  • mlt_2430 acacgctgggcctctctaa R.N. mlt_2431 aaaagggcttatggcagcag R.N. mlt_2432 acagtgggcagggaatgaat R.N. mlt_2433 caaaagcgacatggcaaac R.N. mlt_2434 CCAGCCCCACTCCATTCT R.N. mlt_2435 ctcccaaagtgctgggatta R.N. mlt_2436 aaaatgccttcatccccgtttt R.N.
  • mlt_2456 gggactcagctaagtgcagg R.N. mlt_2457 gggggtctggtaataggggt R.N. mlt_2458 gcagttcagctctcacCTGT R.N. mlt_2459 ggtctgtgggcatttgttg R.N. mlt_2460 cacactcacCGGGCTCAG R.N. mlt_2461 ggagtgaggagcctgtcc R.N. mlt_2462 cttcccaccttccccttct R.N.
  • mlt_2463 aaaatagtgcctgaaaagacga R.N.
  • mlt_2464 gtttcagaatcggtattatattcca R.N.
  • mlt_2465 tttttaattttggaggaatcactta R.N.
  • mlt_2466 gctgaagaacactgtcagctt R.N.
  • mlt_2468 acgtacttacCTTGTGAAAATGC R.N.
  • mlt_2634 caagaacaagggaaacaccaa R.N.
  • mlt_2635 aagaaaagtttgctggagatacat
  • mlt_2636 caaagcaaagcatgtttagtgc
  • mlt_2637 tggtctggcagctataatgaga R.N.
  • mlt_2638 tgaaaacacttcctgcgtttc R.N. mlt_2639 ctctggaccctactccctca R.N. mlt_2640 tctgttcacaaacttcctgtca R.N.
  • mlt_2641 aaggctgtttttcagaaaggt R.N.
  • mlt_2642 cCTGTCAGATCCATTCCAACA R.N.
  • mlt_2643 tccacagagctttctccatgt R.N.
  • mlt_2644 CCTACAGACCGCGCAGAGA R.N.
  • mlt_2645 GGCTACCCCTTCCTCTTCTG R.N.
  • mlt_2646 aacctgtgcccttctctgtc R.N. mlt_2647 ttcgacactttcaacagtaggc R.N.
  • mlt_2648 tgttacaatcatttgccatttc R.N. mlt_2649 CCTGAAGCGCACAGGAG R.N. mlt_2650 tttatgtgacgcccttgatg R.N. mlt_2651 acctgagatctgtgctgtcg R.N. mlt_2652 atgtgccttcccttggactg R.N. mlt_2653 cgatgcgtttagaaggctct R.N. mlt_2654 tgtatcctatggcaggtagtcaga R.N.
  • mlt_2655 ctgacaggtcctgcctatgg R.N. mlt_2656 ggacggggctaaccgaac R.N. mlt_2657 gcagcagactttgtggtca R.N. mlt_2658 ggtgaaaccccatctgtagg R.N. mlt_2659 tgagctttccaatctgctca R.N. mlt_2660 cacgaccctgaggaaggta R.N. mlt_2661 gcagaaaagaagtttctgtgattt R.N.
  • mlt_2682 gtccacgggaaagcacagta R.N. mlt_2683 gccgtatctgtgtaggtatgtg R.N. mlt_2684 aggagttgaccagctttcct R.N. mlt_2685 gaggaaaatgtgactgggaaa R.N. mlt_2686 tcccagttttcttatttttcaga R.N. mlt_2687 cctaaaggtggtcctttgtttg R.N.
  • mlt_2688 acagatgtcccctccttcc R.N.
  • mlt_2689 agttttcatcacaccataatctttta R.N. mlt_2690 TCCATTCAGATGCCTCTCTG R.N.
  • mlt_2691 agatcaagaaactaatgcaagtgg R.N.
  • mlt_2692 CTCCTTAATCTTTGCTCCTTGA R.N.
  • mlt_2693 tgccattatcccaatggtt R.N.
  • mlt_2694 tcattgattctccccgctgc R.N.
  • mlt_2702 catgcattgccacatcac R.N. mlt_2703 gcgtggtgagtgaaatcttg R.N. mlt_2704 ctaggacacaccgcgcttc R.N. mlt_2705 tgattgcttcccagccttt R.N. mlt_2706 caccaaactctgtgtactgacC R.N. mlt_2707 ttacccccaaaccaattatg R.N. mlt_2708 tgaaaataagagtgtgtgaaatacCTGA R.N.
  • mlt_2709 tcattaaaagcattgctctgTC R.N. mlt_2710 ACGAGAGCCAGTCCTCACAG R.N. mlt_2711 gctcacagaagtcaggcatc R.N. mlt_2712 agatgtgggaaatccgtgtc R.N. mlt_2713 GAGGCCAGCACCACAGAC R.N. mlt_2714 GGTCATCCTCTTACTGGACTCG R.N. mlt_2715 gggcgatgactgtcgatgt R.N. mlt_2716 tagtgacttgcaggacaccc R.N.
  • mlt_2731 ccgagaagccggtctgg R.N. mlt_2732 gcagGTGTCCTGCCTGAA R.N. mlt_2733 AGCATCATCAGCATCACAGG R.N. mlt_2734 CTTTGGGGGGTGAGGAAAAGT R.N. mlt_2735 GGCCTGTGCATCTGACTATG R.N. mlt_2736 cTCTTCATTCAAGGCACACC R.N. mlt_2737 ggaagtaaacaaacCTCTTTTGG R.N. mlt_2738 gttacaattgctgccaatgat R.N.
  • mlt_2760 acaggttacacacacacagcccgaac R.N. mlt_2761 gccctgagcccacCTTA R.N. mlt_2762 gccatgccactcacCTGT R.N. mlt_2763 CTCTGCATTGGAGGCTGTG R.N. mlt_2764 cgcaccaagcagacaaagt R.N. mlt_2765 agttccccgtctgcaaaat R.N. mlt_2766 tgcccctaacatcacaatg R.N.
  • the first PCR reaction was performed by adding 0.4 ⁇ l of 10mM dNTP and 2 units of h-Taq DNA polymerase to the template DNA in a thermocycler under the following reaction conditions: once at 95°C; 95°C for 10 seconds, 57°C for 30 seconds, 68°C for 40 seconds 32 times; 68°C for 10 minutes once.
  • Nested PCR used 2ul of DNA amplified in the first PCR as a template and the conditions were the same as the first PCR except for the primers, which were performed by adding primers for nested PCR according to the given conditions.
  • a sequencing library suitable for the Illumine Sequencing platform was created using the nested PCR amplification product, and high-throughput sequencing was performed. carried out.
  • the sequencing library was electrophoresed on a 2% agarose gel and is shown in Figure 2.
  • the ratio of sequences with an intact adapter sequence structure was found to be related to the total amount of primers in the primer pool used to amplify the target DNA sequence.
  • the base sequences were determined through NGS using the DNA library selectively amplified in Example 2, and then bioinformatic base sequence analysis was performed. At this time, NGS was performed by taking the same volume of DNA solution from each library. NGS sequence production according to the total amount of primers used for each primer pool is shown graphically in Figure 3. It can be seen that there is a difference in NGS sequence production depending on the total amount of primers used.
  • the ratio of sequences with an intact adapter sequence structure was independent of the target DNA sequence and the primer pool used to amplify the target DNA sequence. It was found to be related to the total amount of primers.
  • the inclusion rate of more than 80% of a good adapter sequence structure was found in the range of 25 to 400 pmole in the R100 group with the smallest number of targets, but this range was reduced to the range of 100 to 300 pmole in the R600 group with the largest number of targets.
  • sequences with intact molecular markers and strand discriminators only the sequences from which these sequences were removed were divided into those in which two or more identical sequence structures were found and those in which only one was found, and then the sequences with two or more identical sequence structures were used to refer to hg38.
  • the genome was mapped using the BLAST program. At this time, the proportion of base sequences mapped to the genome is shown graphically in Figure 5. As can be seen in Figure 5, it was confirmed that the size of this ratio varies greatly depending on the total amount of primers included in the reaction, similar to what was confirmed in Figures 3 and 4 described above.
  • the amount of base sequences mapped to the target and non-target regions is shown in Figure 6, and a line graph for the target hit rate is also shown. As can be seen in Figure 6, it can be seen that there is a range of the total amount of primers that can produce good mapping quantity and target hit rate depending on each primer pool.
  • Reads mapped to the target region for each library were classified by molecular markers and strand identifiers to construct a molecular duplex consensus sequence.
  • each strand must consist of at least two or more reads, and the duplex consensus satisfies the standard that both sequences composed of the two strands exist.
  • the ratio of base sequences used to construct the molecular double consensus among base sequences with intact adapters was calculated and shown graphically in Figure 8. As this ratio can also be seen in Figure 8, it was confirmed that the range of good results for each primer pool was limited depending on the total amount of primers.
  • the conditions for capturing at least 80% of targets of 5 or more duplex sequences were the total amount of primers in the range of 25 to 300 pmole for the R100 group, and the range was reduced to 100 to 200 pmole for R600 with 600 target sites. I was able to confirm.
  • the conditions for using primers suitable for selective amplification of multiple targets are that the total amount of primers is in the range of 25 to 300 pmole and the amount of individual primers is in the range of 0.167 to 1.0 pmole under the condition of 20ul of PCR reaction solution. there is.
  • duplex consensus sequencing was performed on 1,244 targets in the primer range of 150 to 300 pmole. As can be seen in Figure 12, more than 90% By achieving a good target capture rate, it was confirmed that the quantitative conditions of the primers suitable for selective amplification of multiple targets worked well even for expanded targets.

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Abstract

Selon un mode de réalisation de la présente invention, un procédé d'amplification d'ADN cible multiple et un procédé de séquençage d'ADN cible, selon la présente invention, permettent une amplification efficace d'une séquence cible dans un échantillon ayant une complexité de séquence élevée, telle qu'un génome ou un transcriptome, et permettent à un marqueur moléculaire et à un marqueur de brin d'être situés dans un adaptateur utilisé dans l'amplification, et l'invention concerne ainsi un procédé capable d'identifier précisément et précisément la structure de séquence d'origine d'une séquence cible par séquençage à haut débit ultérieur et analyse bioinformatique d'un produit d'amplification.
PCT/KR2023/013102 2022-09-01 2023-09-01 Composition pour amplification sélective d'adn cible multiple, et procédé d'amplification l'utilisant WO2024049276A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20170142871A (ko) * 2016-06-16 2017-12-28 한국 한의학 연구원 돌연변이 발생률의 측정 방법
KR20190116773A (ko) * 2018-04-05 2019-10-15 한국 한의학 연구원 분자 인덱스된 바이설파이트 시퀀싱
KR20200138183A (ko) * 2018-01-29 2020-12-09 세인트 쥬드 칠드런즈 리써치 호스피탈, 인코포레이티드 핵산 증폭을 위한 방법
KR20220011725A (ko) * 2019-06-26 2022-01-28 엠쥐아이 테크 컴퍼니 엘티디. 네스티드 다중 pcr 고처리량 시퀀싱 라이브러리의 제조 방법 및 키트
WO2022055984A1 (fr) * 2020-09-08 2022-03-17 Resolution Bioscience, Inc. Adaptateurs et procédés de construction à haut rendement de bibliothèques génétiques et d'analyse génétique

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20170142871A (ko) * 2016-06-16 2017-12-28 한국 한의학 연구원 돌연변이 발생률의 측정 방법
KR20200138183A (ko) * 2018-01-29 2020-12-09 세인트 쥬드 칠드런즈 리써치 호스피탈, 인코포레이티드 핵산 증폭을 위한 방법
KR20190116773A (ko) * 2018-04-05 2019-10-15 한국 한의학 연구원 분자 인덱스된 바이설파이트 시퀀싱
KR20220011725A (ko) * 2019-06-26 2022-01-28 엠쥐아이 테크 컴퍼니 엘티디. 네스티드 다중 pcr 고처리량 시퀀싱 라이브러리의 제조 방법 및 키트
WO2022055984A1 (fr) * 2020-09-08 2022-03-17 Resolution Bioscience, Inc. Adaptateurs et procédés de construction à haut rendement de bibliothèques génétiques et d'analyse génétique

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