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WO2018171637A1 - Détection d'une mutation de gène rare - Google Patents

Détection d'une mutation de gène rare Download PDF

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WO2018171637A1
WO2018171637A1 PCT/CN2018/079880 CN2018079880W WO2018171637A1 WO 2018171637 A1 WO2018171637 A1 WO 2018171637A1 CN 2018079880 W CN2018079880 W CN 2018079880W WO 2018171637 A1 WO2018171637 A1 WO 2018171637A1
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primer
amplification
template
round
blocker
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PCT/CN2018/079880
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Chinese (zh)
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陈汉奎
刘和录
覃海德
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陈汉奎
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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
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • This paper deals with the detection of gene mutations, especially the method of selectively enriching rare gene mutations, the corresponding enrichment system, primers and hinders in the enrichment system.
  • a gene mutation is a phenomenon in which a base sequence of a genomic DNA molecule is mutated.
  • a gene mutation refers to a change in the base pair composition or arrangement order of a gene.
  • the research of gene mutation has become one of the hotspots in life science research, and the detection method has also developed rapidly. Especially the establishment of Next Generation Sequencing (NGS) has made the gene mutation detection enter a high-throughput. The new era.
  • NGS Next Generation Sequencing
  • Rare gene mutations refer to extremely rare genetic mutations that are present in the context of a large number of wild gene sequences.
  • the most common example of rare gene mutations is the low-frequency genetic variation that occurs in tumor cells.
  • Many somatic mutations that cause tumors are doped in wild-type cells, and the resulting DNA samples carry a large amount of wild-type DNA.
  • the content of gene mutations is often below 10% or lower.
  • ctDNA circulating DNA
  • ctDNAs carry the same genetic mutation information as in tumor cells, and can also be used for early detection, targeted drug use, and prognosis testing of tumors.
  • ctDNA is non-invasive sampling, and it can reflect the whole picture of tumor gene mutation from the mutation information. Therefore, the liquid biopsy technology with ctDNA as the detection object has its superiority, and its application has been paid more and more attention.
  • Targeting NGS technology is currently the most recognized technology for detecting ctDNA. Due to the low ctDNA content, targeting NGS requires selective design of detection site panels, and the use of probe capture or Ampliseq amplification techniques to enrich and build the test area to improve data efficiency. However, for low-frequency mutations, these enrichment methods do not improve the sensitivity of detection, and can only rely on deepening the sequencing depth (the number of reads of the same template) to complete the detection of rare mutations. For example, for a 0.1% mutation, a sequencing depth of 10,000x or more is required to detect it. NGS faces challenges for mutations below 0.1% due to sequencing chip capacity and cost constraints. How to further increase the ratio of rare mutations to reduce the dependence on sequencing depth is the key to improve the sensitivity of NGS and reduce the cost of sequencing.
  • PCR polymerase chain reaction
  • the PCR method described herein includes steps of denaturation, primer annealing, and extension in the first round of amplification, but does not contain a blocker; in the second round of amplification, in turn includes denaturation, Block annealing, primer annealing, and extension steps.
  • the invention also provides a method for designing PCR primers and hinders, comprising: designing corresponding forward primers and reverse primers according to target gene mutation sites, the forward primers are located upstream of the mutation sites, and the reverse primers are located Downstream of the mutation site; and, by designing a blocker according to the target gene mutation site, the blocker selectively binds to the wild type template to prevent binding of the primer to the wild type template, thereby inhibiting amplification of the wild type template.
  • the blocker sequence covers a genetic mutation site.
  • the 5'-end of the blocker overlaps with the 3'-end of the forward primer or with the 3'-end of the reverse primer, and the blocker bound to the template can prevent annealing of the primer to the template through this overlap region.
  • the 3'-end of the blocker is blocked by conventional techniques such that the template bound to the blocker is unable to undergo an extension reaction of PCR.
  • the downstream reverse primer whether it is a forward primer or a reverse primer, does not cover the mutation site; the barrier is covering the mutation site, but is paired with the wild type of the site, and Mutations at the site cannot be paired, and the blocker overlaps at the 5'-end with the 3'-end of the forward or reverse primer for the same gene mutation, allowing the binding of the blocker to the wild-type template to prevent overlap
  • the primer binds to the same template, and the blocker is blocked at the 3'-end, resulting in failure to be extended.
  • PCR amplification system in which a combination of primers and hinders as described above is made for each gene mutation.
  • the polymerase in the system is a high fidelity DNA polymerase.
  • the primers and hinders for the same gene mutation described herein have the following Tm values: melting temperature (Tm-BW) > (above) primers after binding of the blocker to the wild type template
  • the melting temperature (Tm-Po or Tm-Pn) of the template is > (higher than) the melting temperature (Tm-BM) after binding of the hindrance to the mutant template.
  • the Tm-BW is about 2 ° C or more above Tm-Po or Tm-Pn.
  • the Tm-BW is 3 ° C or more above the Tm-BM.
  • the melting temperature (Tm-Pn) of each of the non-overlapping primers is not lower than the melting temperature (Tm-Po) of the corresponding overlapping primers.
  • Also provided herein is a method of detecting a rare gene mutation using any one or more of the primer-block combinations described herein to perform any of the PCR methods described herein.
  • pairs of forward and reverse primers were designed based on the upstream and downstream sequences of each mutation site.
  • the sequences of these primers do not cover the site where the mutation is located.
  • Each primer typically has a length of no more than 30 bases, preferably no more than 25 bases, more preferably no more than 20 bases.
  • the paired forward and reverse primers result in a sequence size of 60 to 200 base pairs (bp), more preferably 60 to 120 bp, still more preferably 60 to 90 bp, for maximally efficient use of the template, for example Free DNA (cf-DNA) template or ctDNA.
  • the concentration of the paired positive and negative primers in the PCR system is the same or substantially the same as each other, for example, the concentration of the reverse primer is within 100% ⁇ 50% of the concentration of the forward primer, more preferably within 100% ⁇ 20%.
  • the concentration of the forward primer or the forward primer is within 100% ⁇ 50% of the concentration of the reverse primer, more preferably within 100% ⁇ 20%.
  • the more than one pair of forward and reverse primers may be more than one pair of forward and reverse primers as described above to amplify different mutation sites on the same template. This can improve sample utilization.
  • the more than one pair of forward and reverse primers do not interfere with each other, and the concentrations in the PCR system are the same or substantially the same, and the meaning of "substantially the same” is as described above.
  • a blocker is designed based on the target mutation site.
  • the sequence of the blocker covers the mutation site and is paired with the wild type at the mutation site, and mismatched with the mutant.
  • the 5'-end of the blocker has an overlap with the 3'-end of the forward primer or the 3'-end of the reverse primer, for example, 1 to 10 bases overlap, more preferably 2 to 10 bases overlap, still more preferably 2 to 6 bases overlap.
  • the overlapping region is at the 5'-most end of the blocker and/or at the 3'-most end of the primer.
  • the blocker bound to the template can block the annealing of the primer with the overlap region through the overlap region. Therefore, the blocker can be considered as a special form of primer.
  • the site of the mutation is covered by the sequence of the blocker, but is outside the overlap zone.
  • the blocker is in the same direction as the primer having the overlap region.
  • the 3'-end of the blocker is closed so that the blocker does not extend after it is combined with the template.
  • Techniques for end closure are known in the art. For example, using a 3'-C3 carbon arm or a 3'-phosphate group to block the 3'-hydroxyl necessary for the PCR extension reaction, or a reverse ligation with another entire nucleotide such that both ends of the entire sequence are 5' - End, and so on.
  • the melting temperature is lowered.
  • the melting temperature (Tm-BW) of the blocker after binding to its corresponding wild-type template is higher than the melting temperature (Tm-BM) of the blocker and the mutant template by more than 3 °C, for example, high. It is 3 to 16 ° C, preferably 3 to 12 ° C higher, more preferably 3 to 10 ° C higher. In this way, the temperature at which the resist is annealed to the wild type template is not annealed to the mutant template.
  • the length and position of the blocker can be adjusted according to the GC content of the blocker so that the Tm-BWs of the different blocks are the same or close to each other ( ⁇ 2° C.), thereby ensuring the temperature at the block annealing temperature in the multiple reaction system. All blocks can anneal to their corresponding wild-type template.
  • the blocker described herein is a linear blocker.
  • the annealing temperature is the onset temperature at which the single strand begins to complement each other to form a double strand.
  • the reaction system has both a template DNA which is melted into a single strand, and a primer which is itself a single strand. Since the template DNA is much more complex than the primer, the collision binding opportunity between the primer and the template is much higher than the collision between the complementary strands of the template, so the probability of the primer pairing with the template complementary greatly exceeds the probability that the template and the template complement each other.
  • primers may also non-specifically bind to other primers or to an unrelated template during random collisions.
  • the ideal annealing is to maximize the pairing of the single-stranded template with the primer, to reduce the complementary pairing of the single-stranded template with the single-stranded template, and the non-specific binding of the primer to the primer or primer to the unrelated template.
  • the block annealing temperature refers to the temperature at which the blocker anneals to the wild type template
  • the primer annealing temperature refers to the temperature at which the upstream and downstream primers in the paired primer both anneal to the template.
  • the annealing temperature of the barrier is higher than the annealing temperature of the primer, so that the inhibitor can anneal to the wild-type template at the annealing temperature of the inhibitor, and the primer It is not possible to anneal to the template; then, when the temperature is lowered to the primer annealing temperature, the region of the wild-type template that binds to the primer is blocked by the blocker, and only the mutant template has a region capable of binding to the primer and thus annealing.
  • Tm Melting temperature
  • Tm is a reference for annealing temperature.
  • Tm is the temperature at which 50% of the primers (or the blockers described herein) and the complementary sequences behave as double-stranded DNA molecules.
  • Tm is determined by fragment length and base composition. The longer the fragment and/or the higher the GC content, the higher the Tm value required.
  • the Tm value can be determined using a computer program such as OligoAnalyzer 3.1 (Integrated DNA Technologies, Inc.). Depending on the formula used and the sequence of the primers, there may be large differences in Tm.
  • the annealing temperature of the primer is generally within 10 ° C of the Tm of the primer, for example, within 5 ° C, or even 1 to 2 ° C lower. Higher annealing temperatures reduce the formation of primer dimers and non-specific products.
  • Tm-BW melting temperature after binding of the blocker to the wild type template.
  • Tm-BM melting temperature after binding of the hindrance to the mutant template.
  • Tm-Po The melting temperature of the primers with overlapping regions combined with the template.
  • Tm-Pn melting temperature of primers without overlap region combined with template.
  • Tm-BW is higher than Tm-BM by more than 3 ° C, preferably by 3 to 16 ° C, more preferably by 3 to 12 ° C.
  • Tm-BW is Tm-Po and/or It is 2 ° C or more higher than Tm-Pn, preferably 2 to 16 ° C higher, more preferably 2 to 10 ° C higher, more preferably 2 ° C higher than 5 ° C, more preferably 2 to 4.5 ° C higher, still more Preferably, it is 2 to 4 ° C higher, and still more preferably 2 to 3 ° C higher, so that at the temperature at which the resist is annealed to the wild type template, neither of the primers is annealed to the template; both Tm-Po and Tm-Pn are higher than
  • the primer annealing temperature described herein is, for example, 1 to 10 ° C higher, preferably 3 to 10 ° C higher, more preferably 3 to 6 ° C higher
  • the order of the Tm values of the primers and the hindrance is from Tm-BW>Tm-Po>Tm-BM and Tm-BW>Tm-Pn>Tm-BM. Therefore, when the temperature of the reaction system decreases from the high denaturing temperature, the temperature at which the resist is annealed to the wild type template is first reached, so that the hindrance anneals to the wild type template, leaving the single-stranded template as a mutant template, and then reaching the positive The temperature at which the reverse primer anneals to the template causes the forward and reverse primers to anneal to the single-stranded mutant template. When the temperature is raised to the extension temperature, the wild type template is blocked by the hindrance and cannot be extended. Only the mutant template Without being blocked by the barrier, it can be extended, and as a result, the mutant template is amplified.
  • Tm when Tm is compared, it refers to the comparison between Tm determined by the same method.
  • the gene amplification methods described herein, particularly, for example, PCR methods, generally involve two rounds of amplification.
  • the upstream forward primer and the downstream reverse primer are used, and the gene fragment including the mutant site and the gene fragment including the corresponding wild type site are amplified indiscriminately, so that the first round After PCR amplification, both wild-type and mutant types were amplified.
  • the second round of amplification uses the product of the first round of amplification as a template. More preferably, two rounds of amplification are performed continuously.
  • the hindrance is introduced into the amplification system before or at the beginning of the second round of amplification, and the difference between the Tm values of the primer and the hindrance is used to set two different annealing temperatures, resulting in the combination of the primer and the template. Blocking, thereby selectively amplifying the mutant sequence. This greatly increases the ratio of rare mutant fragments in the amplified product. More specifically, for each mutation, there is only one annealing temperature for the primer in the first round of PCR, and PCR amplification is not selective for wild-type and mutant templates; in the second round of PCR, there are two annealing temperatures.
  • One is for annealing of the barrier (specifically, the resist is annealed to the wild type template), and the other is for the annealing of the primer, and the annealing temperature of the barrier is higher than the annealing temperature of the primer, so that the second round of PCR is selective to the template, only the mutation
  • the type is amplified. Since the annealing step in the PCR reaction is generally carried out by lowering the temperature to the annealing temperature from high temperature denaturation, in the second round of amplification of the PCR method described herein, as the reaction temperature decreases from the denaturation temperature, it first drops to the resistance.
  • the annealing temperature specifically the temperature at which the resist is annealed to the wild type template, is then further lowered to the primer annealing temperature. That is, in the second round of amplification, the block annealing is preceded by primer annealing.
  • the second round of amplification steps are followed by denaturation, block annealing, primer annealing, and extension; the corresponding first round of amplification steps are followed by denaturation, primer annealing, and extension.
  • the block annealing means that the blocker is annealed to the wild type template;
  • the block annealing temperature refers to the temperature at which the blocker anneals to the wild type template.
  • the blocker could not bind to the mutant template due to mismatch with the mutant base.
  • the primer overlapping with the blocker can bind to the mutant template, initiate the PCR reaction and extend, thereby causing amplification of the mutant.
  • the blocker is added to the reaction system after the first round of amplification of each cycle, either immediately before the second round of amplification or at the beginning of the second round of amplification.
  • the inhibitor can bind to the template but cannot participate in the extension reaction, so the amount and concentration thereof do not substantially change in the reaction system.
  • the hindrance is excessive for the wild type template to ensure its inhibition efficiency.
  • the amount of the inhibitor may be a multiple of the product of the initial content of the template in the reaction system multiplied by the number of amplification cycles of the first round, for example, 1 to 1000 times, preferably 10 to 1000 times, more preferably 10 to 500 times the product. It is more preferably 10 to 100 times.
  • the concentration of the inhibitor used in the PCR system is substantially the same as the concentration of the corresponding primer, for example, 1 to 4 times, preferably 1 to 2 times.
  • both the first round and the second round of amplification employ a high fidelity DNA polymerase.
  • the number of cycles of the first round and the second round of amplification may be from 1 to 45, respectively.
  • Those skilled in the art can vary depending on the nature, concentration and content of the original template and the amount of the inhibitor, and even combine two rounds of amplification into one round of amplification containing the hindrance.
  • the PCR reaction system described herein may include a combination of a pair of primers and a blocker for each gene mutation (referred to as "primer-blocker combination"). ). Accordingly, the first round and/or the second round are amplified as multiplex PCR, and two or more pairs of positive and negative primers are used to amplify different rare mutation sites.
  • the PCR method described herein particularly shows its advantages of simple operation and cost advantages, and only needs to design a pair of primers and a blocker for each mutation. Selective mutant amplification can be achieved without complex reaction systems or reaction conditions, and the dependence of NGS detection on sequencing depth can be reduced.
  • the PCR methods described herein can be used to detect rare gene mutations, particularly rare mutations in cfDNA or ctDNA, such as tumor gene mutations. Accordingly, the sample detected by the methods herein is preferably a sample containing cfDNA or ctDNA.
  • the ctDNA content in plasma is extremely low, even in some early tumor patients, even less than 0.1%, often determining the presence of such rare mutations needs to reach 10,000X or Higher sequencing depth; secondly, the background noise of NGS sequencing is very high, and the tumor mutation signal is completely submerged in the background noise by the conventional database sequencing method.
  • the technical solutions described herein have unique advantages in solving these two problems.
  • the ratio of the target sequence in the entire sequencing library can be significantly increased, thereby effectively reducing the background noise and avoiding the false negative result of NGS.
  • the ratio of rare mutations to wild-type is significantly increased, for example, the sensitivity to rare mutations is increased to 0.001% or even lower, thereby greatly reducing the requirement for sequencing depth.
  • the method described herein also significantly reduces the cost of sequencing and analysis while improving detection sensitivity and accuracy, and has obvious advantages in clinical application of tumor liquid biopsy.
  • the rare mutation sites detected by the methods herein may be somatic mutation sites of tumor genes, such as SNV and Indel.
  • a gene amplification method in particular a PCR method, comprising:
  • the second round of amplification comprises two annealing temperatures, a block annealing temperature and a primer annealing temperature, and the block annealing temperature is higher than the primer annealing temperature.
  • annealing temperature of the inhibitor is higher than the annealing temperature of the primer by 2 ° C or higher, preferably 2 ° C or more but less than 5 ° C, more preferably 2 to 4.5 ° C higher, still more preferably 2 higher. It is more preferably ⁇ 4 ° C higher than 2 to 3 ° C.
  • the amount of the inhibitor is a multiple of a product of the initial content of the template in the reaction system multiplied by the number of amplification cycles of the first round, for example, 1 to 1000 times the product. It is preferably 10 to 1000 times, more preferably 10 to 500 times, still more preferably 10 to 100 times.
  • a combination of a PCR primer and a hindrance characterized in that each of the gene mutation sites has a forward primer, a reverse primer and a hindrance, wherein:
  • the forward primer corresponds to the upstream sequence of the mutation site
  • the reverse primer corresponds to the downstream sequence of the mutation site
  • the two primers themselves do not cover the mutation site
  • the blocker covers the mutation site and is paired with the wild type at the mutation site, unable to pair with the mutant, the 3'-end of the blocker is blocked, the 5'-end of the blocker and the 3'-end of the forward primer or There is overlap with the 3'-end of the reverse primer.
  • index value range fluctuates by 50%, preferably by 20%, more preferably by 15%, more preferably by 10%.
  • this paper deals with a method for highly selective amplification of rare gene mutations, which inhibits wild-type amplification by specific inhibitors and achieves the goal of enriching rare mutant products, thereby enabling efficient detection of rare mutations.
  • the gene mutation detection method herein has high specificity, and the resistance pair against the wild type template can tolerate a high copy number wild type template.
  • the method described in the present invention can simultaneously detect a single-digit copy of a mutant template of multiple sites, for example, a template having a mutation content of 1/10000 can be effectively detected, which has high sensitivity; and can also be targeted to specific rare Mutations, such as 0.1% rare mutations, are efficiently detected with a smaller sequencing depth (e.g., 100 times smaller) than in the prior art.
  • the gene detection method described in the present invention is aware of a site susceptible to mutation in the wild-type gene, the detection can be performed without knowing the sequence of the gene mutation in advance, and therefore, the technique described herein can simultaneously detect the coverage in the barrier region.
  • a number of known mutation sites can also reveal new gene mutation forms at or near known mutation sites.
  • the present invention is also applicable to the detection of other gene mutations other than those described herein, and also to other application platforms for gene detection such as genotyping.
  • the specific embodiments of the technical solutions described herein are described in detail below, and they should not be construed as limiting the scope of the invention.
  • Figure 1 shows an illustrative example of the PCR described herein.
  • the schematic diagram adopts a system in which the first round of amplification is 20 cycles and the second round of amplification is 15 cycles.
  • the first round of PCR contains only the forward and reverse primers and the DNA template
  • the long black solid line indicates the target DNA fragment
  • the "X" indicates the rare mutation position
  • the single-headed arrow " ” indicates the primer.
  • Figure 1B shows that both the wild type and the mutant are amplified after the first round of PCR amplification.
  • Figure 1C shows that after the first round of PCR, the blocker with the 3' end is added, and the loop is The short black solid line indicates the hindrance.
  • the blocker is stably bound to the wild type template because the primer has a low Tm value, and the primer cannot bind to the template. Then, the primer is annealed. At the temperature, the blocker that has stably bound to the wild-type template inhibits the binding of the forward primer to the wild-type template by the base whose 5' end overlaps with the 3' end of the forward primer, thereby preventing the expansion of the wild-type sequence. However, because the blocker cannot bind to the mutant template, the blocker cannot prevent the binding of the forward primer to the mutant template, and the mutant sequence is amplified.
  • the long black solid line indicates the target DNA fragment
  • "X "" indicates a rare mutation position indicating that the mutant template was selectively amplified after the second round of PCR.
  • Figure 2 shows the temperature and cycle parameter settings for PCR amplification.
  • the first round of PCR uses 20 cycles
  • the second round of PCR uses 15 cycles.
  • There was only one annealing temperature for the primer in the first round of PCR and amplification of the PCR was not selective for the wild type template and the mutant template.
  • the second round of PCR there is block annealing and primer annealing, and the annealing temperature of the block is higher than the annealing temperature of the primer, so that the second round of PCR is selective to the template, and only the mutant template is amplified.
  • Figure 3 shows that the blocker effectively inhibits the expansion of the wild type template.
  • This example shows the change in Ct values before and after the addition of the EGFR-Ex19del wild type template to the inhibitor.
  • the Cycle threshold represents the number of cycles experienced by the fluorescent signal within each reaction tube when it reaches a set threshold.
  • ⁇ Rn represents the fluorescence signal after subtracting the background signal from the reaction tube.
  • the ⁇ Rn threshold is generally controlled within the exponential growth phase of the amplification curve, and the Ct value is determined by the intersection of the ⁇ Rn threshold line and the amplification curve.
  • the ⁇ Rn threshold line is 5000
  • the amplification is carried out according to 2n, and the number of starting templates of the two tubes is the same, and the reaction tube containing the inhibitor has used 9 cycles to reach the ⁇ Rn threshold line of 5000, thereby showing the expansion of the reaction tube containing the inhibitor.
  • the figure also shows that the amplification curve containing the inhibitor is no longer a typical S-shaped curve due to the hindrance of the inhibitor.
  • Figure 4 shows that the blocker does not affect the amplification of the mutant template.
  • the amplification curve maintains a typical S-shaped curve.
  • a corresponding upstream forward primer, a downstream reverse primer, and a hindrance covering the mutation site are designed for the target gene mutation site to be detected.
  • the upstream and downstream primers are not selective for wild type and mutant types.
  • the blocker is designed for the wild type and has a mismatch with the mutant template. Blocking the 5'-end and 3'-end of the upstream primer was overlapped 2-6 nucleotides (nt), the 3'-end thereof is closed barrier PO 4 group.
  • annealing temperature (54 ° C) for the primer in the first round of PCR is 1 to 5 ° C lower than the melting temperature (Tm-Po) of the forward primer, and the annealing time is 20 s;
  • There are two annealing temperatures in the PCR one is 58 ° C (time 30 s), the resistance is annealed, and the other is 54 ° C (time 20 s), the same as the first round, for primer annealing.
  • the blocker is selective to the template, and can stably bind to the wild type template and prevent its amplification, and finally only the mutant type is amplified.
  • Blockers were added to the PCR system containing the homozygous wild-type template, and the difference in Ct values was compared with PCR without addition of the inhibitor to observe the efficiency of the inhibitor.
  • Figure 3 shows the results: compared with the PCR amplification without the inhibitor, the Ct values of the four wild type templates after adding the inhibitors increased to different extents, and the negative control without the template did not show the amplification curve.
  • the PCR results showed that the inhibition efficiency of the four kinds of inhibitors to their corresponding wild-type templates was between 100-500 times.
  • the experimental results are consistent with the theoretical speculation, indicating that the technique has high specificity, that is, the amplification of the wild type template is effectively inhibited.
  • a cfDNA-positive standard containing the above 4 mutations at a frequency of 0.1% was used (not due to the biological company, product number HD780, which contained 0.1% rare gene mutation and 100% wild).
  • the template was used as a template, and two rounds of multiplex PCR amplification were performed according to the above PCR system. The first round uses 20 cycles and contains no obstructions. The second round used 15 cycles and contained 400 nM of blocker (the control contained no blockers).
  • the PCR product was subjected to purification and database construction, and finally sequenced on the DA8600 platform (Daan gene) and compared with the control without the inhibitor.
  • the present invention employs a mutation site-specific blocker to prevent amplification of the wild-type template and achieve enrichment of rare mutations in the template, thereby detecting rare mutations with high selectivity.
  • the hundreds-fold enrichment of rare mutations not only significantly improves the detection sensitivity, but also greatly reduces the requirement for NGS sequencing depth, thus greatly reducing the cost of NGS sequencing.
  • the present invention is also applicable to the detection of other gene mutations other than the present embodiment, and is also applicable to other application platforms for gene detection such as genotyping. Therefore, the scope of protection of the present invention is not limited to the embodiments.

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Abstract

L'invention concerne un procédé de détection d'une mutation de gène rare, comprenant les étapes consistant à : utiliser une amorce et un répresseur de détection de mutation de gène hautement sélectifs, et amplifier le type sauvage par inhibition spécifique.
PCT/CN2018/079880 2017-03-23 2018-03-21 Détection d'une mutation de gène rare WO2018171637A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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