WO2017088750A1 - Procédé et kit pour l'amplification de déviation d'une séquence d'acide nucléique cible dans un échantillon - Google Patents

Procédé et kit pour l'amplification de déviation d'une séquence d'acide nucléique cible dans un échantillon Download PDF

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WO2017088750A1
WO2017088750A1 PCT/CN2016/106872 CN2016106872W WO2017088750A1 WO 2017088750 A1 WO2017088750 A1 WO 2017088750A1 CN 2016106872 W CN2016106872 W CN 2016106872W WO 2017088750 A1 WO2017088750 A1 WO 2017088750A1
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nucleic acid
sequence
target nucleic
acid sequence
temperature
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PCT/CN2016/106872
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English (en)
Chinese (zh)
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葛猛
王宏伟
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葛猛
北京福安华生物科技有限公司
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Priority to CN201710304210.3A priority Critical patent/CN107354196B/zh
Priority to CN201710304209.0A priority patent/CN107354195B/zh
Priority to CN201710304254.6A priority patent/CN107354197B/zh
Publication of WO2017088750A1 publication Critical patent/WO2017088750A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present application relates to the field of molecular biology.
  • the present application relates to methods and kits for bias amplification of a target nucleic acid sequence in a sample.
  • the inventors of the present application have considered the problem of detecting low frequency gene mutations in the field, and have carried out extensive research to realize bias amplification of nucleic acid molecules containing low frequency mutations, and the nucleic acid molecules subjected to bias amplification can be used for subsequent multiplication.
  • a detection method to identify low frequency gene mutations is a detection method to identify low frequency gene mutations.
  • the present application provides a method for bias amplification of a target nucleic acid sequence in a nucleic acid sample, the target nucleic acid sequence having one or more mutations relative to its wild-type form, the method being a method A or method B; the method A may include the following steps in sequence:
  • the denaturation step comprises:
  • Tc critical denaturation temperature
  • melting temperature of the duplex forming temperature than the second wild-type form a closed sequence of the target nucleic acid sequence (T m) Low 1-5 ° C, and higher than the T m of the wild-type duplex of the target nucleic acid sequence;
  • the method B may include the following steps in sequence:
  • step ( 2 ) of the method A
  • the method described above further comprises the step (3) of isolating and purifying the amplification product obtained in step (2).
  • Techniques for isolation and purification of PCR amplification products are well known to those skilled in the art.
  • the amplification product mixture can be fractionated by agarose gel electrophoresis, and the bands at the molecular weights can be subjected to nucleic acid separation and purification according to the length of the target nucleic acid sequence.
  • nucleic acid sample refers to a mixed sample of nucleic acid molecules, including, but not limited to, genomic nucleic acid samples or cDNA nucleic acid samples presented from tissues or cells.
  • target nucleic acid sequence is a nucleic acid fragment having one or more mutations relative to its wild-type form relative to a "non-target nucleic acid sequence".
  • target nucleic acid sequence The column can be a fragment of a gene coding region as well as fragments of other functional regions, for example, a promoter, an intron, an enhancer, and the like.
  • the non-target nucleic acid sequence can comprise a target nucleic acid sequence that has not been mutated, i.e., a wild-type form.
  • the inventions of the present application are particularly suitable in the case where the "target nucleic acid sequence" is lower in the ratio of the sample (i.e., the target nucleic acid sequence / (target nucleic acid sequence + target nucleic acid sequence wild type)).
  • the ratio of the target nucleic acid sequence in the sample is from about 1 to 5%, such as from about 1%, 2%, 3%, 4% or 5%, and the like.
  • the target nucleic acid sequence is less than or above the above range or value for all nucleic acid molecules in the nucleic acid sample.
  • biasing amplification as used herein is also understood to mean “enriched amplification”, meaning that the degree of amplification of a target nucleic acid sequence is greater than that of a non-target nucleic acid sequence (eg, a wild-type target nucleic acid sequence). The degree of increase is such that the target nucleic acid sequence is prominently displayed in the nucleic acid sample.
  • the original ratio of the target nucleic acid sequence is 1%, and the original ratio of the non-target nucleic acid sequence is 99%; after "biased amplification", the proportion of the target nucleic acid sequence becomes 20%, instead of The original ratio of the target nucleic acid sequence is 80%, and then the target nucleic acid sequence is 20 times enriched.
  • wild type refers to the most common form of a nucleic acid molecule in its natural state.
  • wild-type nucleic acid refers to a nucleic acid molecule that does not undergo any mutation.
  • the target nucleic acid sequence comprises about 40-250 bases, such as about 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170 , 180, 190, 200, 21 0, 220, 230, 240, 250 bases, etc., or any range of the above values.
  • the target nucleic acid sequence may also comprise about more or fewer bases.
  • a single-stranded closed sequence complementary to the sense strand and/or the antisense strand of at least a portion of the wild-type form of the target nucleic acid sequence is provided, wherein the closed sequence comprises one or more locked nucleic acids Replacement.
  • the wild-type sequence of the target nucleic acid sequence is generally obtainable, and thus, the sequence of the single-stranded closed sequence complementary to its sense strand and/or antisense strand is also obtainable.
  • techniques for introducing a lock nucleic acid substitution in the synthesis of a sequence are also known.
  • the single-stranded blocking sequence is complementary to the sense strand and/or the antisense strand of at least a portion of the wild-type form of the target nucleic acid sequence.
  • the single stranded closed sequence is complementary to the sense strand and/or the antisense strand portion of the target nucleic acid sequence.
  • locked nucleic acid substitution refers to the replacement of an original natural nucleomonomer with a locked nucleic acid monomer. For example, for the sequence ⁇ iJ "ACGG”, the "locked nucleic acid substitution" of the second position means replacing the original C with the locked nucleic acid monomer C', and the sequence after replacement is "AC*GG".
  • the blocking sequence comprises from about 10% to about 100% of a locked nucleic acid substitution.
  • about 10% to 100% of the base form of the target nucleic acid sequence is replaced with a locked nucleic acid monomer.
  • the ratio may be about 10 ⁇ 3 ⁇ 4, 15% ⁇ 20%, 25%- 30% ⁇ 35% ⁇ 40%, 45%-50 ⁇ 3 ⁇ 4, 55%, 60%, 65%, 70%, 75 %, 80%, 85%, 90%, 95%, 100%, etc., or any range of the above values. It will be understood by those skilled in the art that since the number of bases contained in a certain nucleic acid sequence is variable, the ratios described above should be understood as approximate guide values and not necessarily exact values.
  • the number of locked nucleic acid substitutions can be understood as 12, which can also be understood as 13; likewise, 50% is 31.5, then the locked nucleic acid is replaced.
  • the number can be understood as 31, which can also be understood as 32.
  • the locked nucleic acid substitutions are substantially evenly distributed in the closed sequence. It will be understood by those skilled in the art that a “substantially" uniform distribution means that the number of unsubstituted bases spaced between each of the locked nucleobases after replacement is substantially the same. In some embodiments, a “substantially” uniform distribution means that the maximum number of unsubstituted bases spaced between each of the locked nucleobases after replacement is no more than three. For example, the number of unsubstituted bases separated by each of the locked nucleobases after replacement is N, and may be at most N+l, N+2, or N+3, or may be all ⁇
  • the locked nucleic acid substitution is designed to have a locked nucleic acid substitution every 2-5 bases, eg, the distribution of the locked nucleic acid substitution is every 2, 3, 4 or 5 There is one replacement base in the replacement base.
  • the length of the blocking sequence is about 20-40 bases shorter than the target nucleic acid sequence, and the ends of the blocking sequence are separate from the forward and reverse primers used in step (2). There is an overlap of approximately 3-5 bases.
  • the blocking sequence comprises a chemical modification at the 3' end that prevents extension of the blocking sequence in a PCR reaction, such as a phosphorylation modification, a C3-spacer modification, a C6-spacer modification.
  • a PCR reaction system comprising the sample, the blocking sequence, and the forward and reverse primers designed according to the target nucleic acid sequence is provided and subjected to a cyclic amplification reaction.
  • a PCR reaction system comprising the sample, the blocking sequence, and the forward and reverse primers designed according to the target nucleic acid sequence is provided and subjected to a cyclic amplification reaction.
  • Methods for forward and reverse primers in PCR amplification reactions, as well as conventional reagents required to perform PCR amplification reactions, are well known to those skilled in the art.
  • the PCR reaction system may also include any one or more of the assays required for the amplification reaction, such as DNA polymerase, dNTP, and the like.
  • PCR reactions are typically multi-cycle amplification (e.g., 20-40 cycles), and each cycle typically includes denaturation, annealing, and extension steps.
  • the denaturation step in the PCR cycle of the present application comprises two phases:
  • the first stage raising the temperature to a sufficiently high temperature to bring the blocking sequence into contact with the nucleic acid sample (the nucleic acid duplex in the nucleic acid sample is capable of cleavage), and then cooling to the first temperature such that the blocking sequence and the target nucleic acid sequence
  • the wild-type form of the target nucleic acid sequence forms a duplex, wherein the first temperature is higher than the annealing temperature of the primer to the template.
  • sufficiently high temperatures e.g., above 95 °C
  • the double stranded nucleic acid will denature and branch into two single strands.
  • the single stranded closed sequence when a single-stranded closed sequence is capable of contacting the sense strand or the antisense strand of the melted target nucleic acid sequence, the single stranded closed sequence is complementary to the sense strand or the antisense strand portion of the target nucleic acid sequence (depending on the closed sequence is directed against
  • the justice chain is also the antisense chain design), and therefore constitutes the structural basis for the combination of double-stranded (closed/target double-stranded).
  • the T m value of the blocking/target double strand will be higher than the 1 1 value of the target/target duplex (ie, the double strand formed by the sense strand and the antisense strand of the original target nucleic acid sequence). Therefore, with the cooling process, the closed/target double strands are preferentially formed. Setting the first temperature to be higher than the annealing temperature of the primer and the template can avoid or reduce the interference of the primer binding on the above process.
  • the cooling process is a natural cooling.
  • the temperature reduction process can also be a controlled temperature drop.
  • the blocking sequence is in excess relative to the target nucleic acid sequence.
  • a second stage the temperature is raised to a second temperature, the second temperature being lower than a melting temperature (T m ) of the duplex formed by the blocking sequence and the wild type form of the target nucleic acid sequence by about 1-5 ° C, and T m is higher than wild-type form a duplex target nucleic acid sequence.
  • T m melting temperature
  • the steps are The closed/target double strand formed in (2) will contain a mismatch; in the same way, since the nucleic acid sample may also contain the wild type form of the target nucleic acid sequence, then The closed/wild-type target duplex formed in step (2) does not contain a mismatch.
  • T m melting temperature
  • T m The melting temperature of the "closed/wild-type target double-strand"
  • the blocking sequence preferentially forms a blocking/target duplex with the target nucleic acid sequence
  • the amount of the blocking/target double strand in the system is much higher than the wild type duplex of the target nucleic acid sequence, which also makes "closed The /target double-stranded "melting" serves as the primary template for binding.
  • step (2) can be implemented as follows:
  • the PCR system is warmed to a sufficiently high temperature (eg, above 95 ° C), and then cooled to a first temperature that is significantly higher than the annealing temperature of the primer and the template, and then raised to a second temperature, in the second At the temperature, the double strand formed between the blocking sequence and the target nucleic acid sequence will preferentially smear; subsequently, the temperature is lowered to the annealing temperature of the primer for annealing; and then the temperature is raised to 72 ° C for extension.
  • a sufficiently high temperature eg, above 95 ° C
  • the present application provides a kit for bias amplification of a target nucleic acid sequence in a nucleic acid sample, the target nucleic acid sequence having one or more mutations relative to its wild-type form, the kit A single-stranded closed sequence comprising a sense strand and/or an antisense strand complementary to at least a portion of a wild-type form of a target nucleic acid sequence, wherein the blocking sequence comprises one or more locked nucleic acid substitutions.
  • the kit further comprises forward and reverse primers designed according to the target nucleic acid sequence.
  • the target nucleic acid sequence comprises between about 40 and 250 bases.
  • the blocking sequence comprises from about 10% to about 100% of a locked nucleic acid substitution.
  • the locked nucleic acid substitutions are substantially evenly distributed in the closed sequence.
  • the length of the blocking sequence is about 20-40 bases shorter than the target nucleic acid sequence, and the ends of the blocking sequence are approximately the same as the forward and reverse primers designed according to the target nucleic acid sequence, respectively.
  • the blocking sequence can be chemically modified.
  • the blocking sequence comprises a chemical modification at the 3' end that prevents extension of the blocking sequence in a PCR reaction, such as a phosphorylation modification, a C3-spacer modification, a C6-spacer modification.
  • the present application provides a system for performing a bias amplification of a target nucleic acid sequence in a nucleic acid sample, the system comprising the closed sequence device of the first aspect, and the method of the first aspect A temperature-controlled amplification device for the P CR amplification reaction.
  • Devices for introducing a lock nucleic acid substitution at a fixed point in a synthetic sequence are commercially available, and many types of PCR instruments currently available can be used in the method described in the first aspect, wherein low requirements for PCR equipment are required. It is also one of the advantages of this application.
  • the application provides a method for detecting a mutation in a target nucleic acid sequence in a nucleic acid sample, the method comprising identifying a mutation in an amplification product obtained according to the method of the first aspect.
  • sequencing can be used, and after the partial amplification, the ratio of the target nucleic acid sequence in the nucleic acid sample is greatly increased to meet the sequencing requirements.
  • probe hybridization may be employed, and of course, sequencing may also be employed.
  • the present application provides a method for detecting a mutation in a target nucleic acid sequence in a sample, comprising the steps of: performing a bias amplification of a target nucleic acid sequence by the method described in the first aspect to obtain an amplification product; Mutations in the amplified product.
  • 1 shows the vector pUC57 for TP53-exon exon clone.
  • 2 shows the results of mass spectrometric identification of four closed sequences.
  • lanes 1-5 are BS60-1
  • lane 6 is the corresponding PCR product PCR-60
  • lanes 7-12 are single-strand BS60-1, BS60-2, BS60-3, respectively.
  • Lane M is DNA Marker DL2000.
  • BS60-1, BS60-2, BS60-3, BS60-4, and RS60 respectively represent products formed by annealing of four closed sequences and reference sequences RS60, respectively, of their reverse complementary order ⁇ ijRS60rc.
  • the melting curve; PCR-60 indicates the melting curve of the corresponding PCR product PCR-60 obtained by amplification.
  • Figure 5 shows the corresponding positions of the different mutant mutation sites in different closed sequences, the underlined letters in the figure indicate the corresponding locked nucleic acid monomers.
  • Figure 6 shows the effect of different single base mismatches on four closed sequences and the reference sequence RS60 Tm values.
  • Figure 8 shows the amplification of pure wild-type and pure mutants by bias amplification at different Tc values.
  • FIG. 9 shows the effect of the amplification condition of the bias amplification.
  • Figure 10 shows the presence of different concentrations of blocking sequences and different Tc values, biased amplification for the same sample (contains
  • Figure 11 shows the enrichment of the same sample (containing the 3% C847T mutation) by bias amplification at different Tc values.
  • the above sequence is the wild type sequence cloned into the vector, wherein the underlined portion is the wild type sequence of the 8th exon of TP53, and the double underlined portion is the amplicon region (87 bp) detected in the following examples.
  • the primers used are as follows:
  • TP8-230F1 5'-CG GGAT CTTACTGCCTCTTGCTTC-3 ' (SEQ ID NO: 2)
  • TP8-230R1 5,-CTC GAG TCTGAGGCAT AACTGCACC-3 ' (SEQ ID NO: 3)
  • the scribed portion shows the cleavage site, and the selected cleavage sites are Sacl and BamH I, respectively.
  • various standard samples can be conveniently prepared by extracting the above several mutant plasmids and diluting them into wild type plasmids in different ratios.
  • Eference Sequence 60 bases, which corresponds to the wild-type form of the target nucleic acid sequence described herein.
  • four different locked nucleic acid sequences were further designed as closed sequences: BS60-1, BS60-2, BS60-3, and BS60-4 (Blocking S
  • RS60, BS60-1, BS60-2, BS60-3 and BS60-4 are as follows:
  • BS60-2 5'-CT C GTGC G *CCGGT C CTCC C *AGGAC A *GGCAC A *
  • BS60-3 5'-CTCTGT G *CGCCGG T *CTCTCCC A *GGACAGGC A *CAAACA C '
  • RS60-4 5'-CT *CTGT G *CG C *CGG T *CTC T *CCCA G *GAC A *GGC A *CAA A *CAC G *CACC T *CA A *AGC T *GTT C *CGT C *C-C3 spacer-3' (3 C*3 G*4A *5 T *) (SEQ ID NO: 12)
  • RS60 and RS60 reverse complement sequence RS60rc are also synthesized.
  • the T m values of the four closed sequences synthesized in Example 2 were determined by the melting curve method.
  • the four blocking sequences and the reference sequence RS60 are mixed with their reverse complementary sequences (RS60 rc) in an equimolar amount, and then placed in a boiling water bath. After high temperature denaturation for 2 minutes, the power is turned off, so that the temperature of the sample follows. The water temperature is lowered and gradually lowered to room temperature, thereby effecting annealing to form the corresponding double strand.
  • the experiment was carried out on a Biomd CFX96 type sputum PCR instrument. The experimental results are shown in Fig. 4. As shown in Fig. 4: The RS60 annealing product was identical to the corresponding PCR product PCR-6 ( ⁇ T m value, again indicating the success of annealing. The experimental results show that the 1 ⁇ values of all four closed sequences are significantly higher than the natural sequence RS60 (the measured values of 1 hail 1 are shown in Table 2), and the number of LNA monomers in the closed sequence is uncomfortable. When it is increased, its Ding will increase.
  • the locked nucleic acid has a stronger affinity for its reverse-complementary DNA sequence relative to the native oligonucleotide sequence. This also makes it possible to adjust the amount of locked nucleic acid in the blocking sequence such that the critical denaturation temperature Tc (below the T m value between the blocking sequence and the target DNA) is slightly higher than the T m value of the PCR product.
  • annealing was carried out at 70 ° C by mixing the excess blocking sequence with the PCR product, denaturation at a high temperature, and then at 70 °C.
  • the experiment used four different blocking sequences and five PCR products including wild type and four mutant types. The position of the LNA monomer in these blocking sequences and the position of the mutation site in the mutant gene are shown in Fig. 5.
  • Example 5 Establishment of a biased amplification platform
  • the blocking sequence preferentially inhibits amplification of the wild type template
  • the method for bias amplification of a target nucleic acid sequence (especially a low frequency mutation in a sample) provided in the present invention provides a basis for the detection of a gene mutation and has important application value.

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Abstract

L'invention concerne un procédé d'amplification de déviation d'une séquence d'acide nucléique cible présentant une mutation par rapport à une forme de type sauvage dans un échantillon, consistant à : utiliser une séquence fermée à simple brin complémentaire à au moins une partie d'un brin sens et/ou d'un brin antisens de la forme de type sauvage de la séquence d'acide nucléique cible, une ou plusieurs substitutions d'acide nucléique verrouillées étant comprises dans la séquence fermée ; utiliser un système de réaction par PCR comprenant l'échantillon, la séquence fermée et une amorce sens et antisens conçue en fonction de la séquence d'acide nucléique cible et effectuer une réaction d'amplification cyclique, les étapes de dénaturation consistant à : élever la température à une température suffisamment élevée, mettre en contact la séquence fermée avec l'acide nucléique dans l'échantillon, puis refroidir à une première température afin que la séquence fermée, la séquence d'acide nucléique cible et la forme de type sauvage de la séquence d'acide nucléique cible forment un duplex, la première température étant supérieure à la température d'hybridation de l'amorce et de la matrice, puis élever la température à une deuxième température inférieure d'environ 1-5°C à la température de fusion (Tm) du duplex formé par la séquence fermée et le type sauvage de la séquence d'acide nucléique cible et supérieure à la Tm du duplex de la forme de type sauvage de la séquence d'acide nucléique cible.
PCT/CN2016/106872 2015-11-25 2016-11-23 Procédé et kit pour l'amplification de déviation d'une séquence d'acide nucléique cible dans un échantillon WO2017088750A1 (fr)

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CN201710304210.3A CN107354196B (zh) 2015-11-25 2017-05-03 一种检测人类kras基因突变的试剂盒
CN201710304209.0A CN107354195B (zh) 2015-11-25 2017-05-03 一种检测人类esr1基因突变的试剂盒
CN201710304254.6A CN107354197B (zh) 2015-11-25 2017-05-03 一种检测人类nras基因突变的试剂盒

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CN201510834275.XA CN107338240B (zh) 2015-11-25 2015-11-25 对样品中目标核酸序列进行偏向扩增的方法和试剂盒

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
CN107164519A (zh) * 2017-06-26 2017-09-15 深圳优圣康医学检验所有限公司 一种基于荧光pcr检测esr1基因突变的引物及探针

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Publication number Priority date Publication date Assignee Title
CN111500688A (zh) * 2020-04-30 2020-08-07 北京和合医学诊断技术股份有限公司 用于同步检测nras基因的第2、3、4号外显子基因突变的方法

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