WO2018133546A1 - Procédé de construction, procédé de détection et kit pour une bibliothèque de détection de mutation de gène de thalassémie alpha fœtale prénatale non invasive - Google Patents

Procédé de construction, procédé de détection et kit pour une bibliothèque de détection de mutation de gène de thalassémie alpha fœtale prénatale non invasive Download PDF

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WO2018133546A1
WO2018133546A1 PCT/CN2017/113231 CN2017113231W WO2018133546A1 WO 2018133546 A1 WO2018133546 A1 WO 2018133546A1 CN 2017113231 W CN2017113231 W CN 2017113231W WO 2018133546 A1 WO2018133546 A1 WO 2018133546A1
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primer set
downstream
upstream
specific
library
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Chinese (zh)
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王晓锋
徐湘民
曾华萍
宋卓
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人和未来生物科技（长沙）有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • 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
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

Definitions

  • the invention relates to the field of gene detection technology, in particular to a method, a detection method and a kit for constructing a non-invasive prenatal fetal alpha thalassemia gene mutation detection library.
  • Thalassemia (referred to as thalassemia) is one of the high incidence genetic diseases in the world. It is due to the mutation or deletion of human genes, resulting in an imbalance of the synthesis rate of ⁇ , ⁇ -globin peptide chains, resulting in hemolytic anemia.
  • Two common types of thalassaemia are ⁇ -thalassemia and ⁇ -thalassemia, ⁇ -thalassemia-related genes are HBA2 and HBA1, and ⁇ -thalassaemia-related genes are HBB.
  • the thalassemia is concentrated in the tropical and subtropical regions, mostly in the Mediterranean countries, followed by the Middle East, India, Pakistan, Southeast Asia, South China and North Africa. The United States is an immigrant country with a high incidence.
  • ⁇ -thalassemia genotypes are commonly found as - ⁇ SEA , - ⁇ 3.7, - ⁇ 4.2, ⁇ CS , ⁇ QS and ⁇ WS , and 26 gene mutations are common in ⁇ -thalassemia. do not. At present, there is no cure for thalassaemia. It can only rely on traditional methods of treatment, that is, blood transfusion therapy is the main method, and the price is expensive. Therefore, prenatal screening and diagnosis are of great significance for preventing such birth defects.
  • Chiu RW achieves efficient detection of the father-derived codons 41/42 4bp deletion by using real-time fluorescent PCR technology, and researchers have developed detection methods for other ⁇ -thalassaemia mutations, but since the detection mutations are mostly point mutations, The specificity of the method needs to be improved. Studies on alpha-thalassemia mutations have also been reported. Warunee Tungwiwat et al. established a method for detecting ⁇ 0 deletions based on real-time PCR, but the sensitivity of this method needs to be improved. In addition, a variety of high-sensitivity techniques have also been tried for non-invasive depletion detection, such as mass spectrometry, digital PCR, COLD-PCR, and the like.
  • the detection methods based on single-site PCR technology have certain limitations, such as low plasma DNA content and high fragmentation characteristics, which may bring false negative PCR.
  • This method can only detect the presence of a specific paternal mutation different from the mother, and cannot further determine the presence or absence of the maternal mutation.
  • Whole-genome sequencing or target region capture sequencing of maternal plasma free DNA based on high-throughput sequencing technology can more accurately detect fetal thalassemia genes, but the cost of genome-wide sequencing and the complexity of analytical methods and the need for parental monomers Types and the like limit the clinical application; target region capture sequencing can only achieve the differentiation of the fetal ⁇ SEA deletion pure heterozygous type due to the low capture efficiency of the target region, and there is also the possibility of typing errors. In view of this, it is necessary to provide a high-precision non-invasive prenatal fetal thalassemia gene mutation detection method.
  • the invention provides a method, a detection method and a kit for constructing a non-invasive prenatal fetal alpha thalassemia gene mutation detection library, which can accurately and efficiently detect the alpha thalassemia gene mutation, and the result is consistent with the amniocentesis detection type, but Safety, non-invasiveness and high efficiency are significantly better than amniocentesis.
  • the present invention provides a method for constructing a non-invasive prenatal fetal alpha-type thalassemia gene mutation detection library for detecting non-invasive prenatal fetal alpha-type thalassemia gene mutation by high-throughput sequencing
  • the above methods include:
  • the upstream specific primer set 1 includes an upstream primer set 1 for amplifying the ⁇ -thalassemia gene and an upstream primer set 1 for calculating a plurality of SNP sites of a fetal free DNA ratio; the above-mentioned downstream specific primer set 1 comprising a downstream primer set 1 for amplifying a thalassemia gene and a downstream primer set 1 for calculating a plurality of SNP sites of a fetal free DNA ratio;
  • the upstream specific primer set 2 includes an upstream primer set 2 for amplifying the ⁇ -thalassemia gene and an upstream primer set 2 for calculating a plurality of SNP sites of a fetal free DNA ratio;
  • the above-mentioned downstream specific primer set 2 comprising a downstream primer set 2 for amplifying the alpha thalassemia gene and a downstream primer set 2 for calculating a plurality of SNP sites of fetal free DNA ratio;
  • the upstream specific primer set 2 is closer to the corresponding amplification target position than the upstream specific primer set 1 described above. Point, the downstream specific primer set 2 is closer to the corresponding amplification target site than the downstream specific primer set 1; the upstream specific primer set 2 and the 5' end of the downstream specific primer set 2 are contained for a linker sequence of a high throughput sequencing library;
  • the upstream specific amplification product 2 and the downstream specific amplification product 2 are mixed, and then amplified using universal primers at both ends to obtain a library which can be sequenced by the machine.
  • the 5' end of the upstream specific primer set 1 and the downstream specific primer set 1 described above are provided with a modification for isolating the amplification product, preferably with a biotin modification.
  • the 3' end of the primer for the specific target site in the upstream specific primer set 1 and the 5' end of the primer corresponding to the target site in the upstream specific primer set 2 are 10-15 bases Coincident; the 3' end of the primer for the specific target site in the downstream specific primer set 1 and 10-15 bases from the 5' end of the primer for the target site in the downstream specific primer set 2 described above Coincident.
  • upstream primer set 1 sequence for amplifying the ⁇ -thalassemia gene is as shown in SEQ ID NOs: 1-7;
  • the upstream primer set 1 sequence of the above plurality of SNP sites for calculating the ratio of fetal free DNA is as shown in SEQ ID NOS: 8-17;
  • the sequence of the downstream primer set 1 for amplifying the alpha thalassemia gene is shown in SEQ ID NOs: 18-23;
  • the above upstream primer set 2 sequence for amplifying the alpha thalassemia gene is shown in SEQ ID NOs: 34-40;
  • the upstream primer set 2 sequence of the above plurality of SNP sites for calculating the ratio of fetal free DNA is shown in SEQ ID NOs: 41-50;
  • the sequence of the downstream primer set 2 for amplifying the alpha thalassemia gene is shown in SEQ ID NOs: 51-56;
  • the sequence of the downstream primer set 2 of the above plurality of SNP sites for calculating the ratio of fetal free DNA is shown in SEQ ID NOs: 57-66.
  • the present invention provides a non-invasive prenatal fetal alpha thalassemia gene mutation detecting method comprising performing high-throughput sequencing of a library constructed by the method of the first aspect to obtain a sequencing read read; Based on the total depth of the unique specific tag sequence of the site and the depth of the allele of the mutation, the fetal DNA content was calculated and the thalassemia-related mutation was typed to analyze the alpha-thalassemia gene mutation.
  • the present invention provides a non-invasive prenatal fetal alpha thalassemia gene mutation detection library construction kit for performing non-invasive prenatal fetal alpha thalassemia gene mutation by high-throughput sequencing
  • the above kits include:
  • upstream specific primer set 1 and downstream specific primer set 1 for upstream pre-library and downstream pre-library, respectively Specific amplification is performed to obtain upstream specific amplification product 1 and downstream specific amplification product 1, respectively.
  • the upstream specific primer set 1 includes an upstream primer set 1 for amplifying the ⁇ -thalassemia gene and an upstream primer set 1 for calculating a plurality of SNP sites of a fetal free DNA ratio; the above-mentioned downstream specific primer set 1 comprising a downstream primer set 1 for amplifying a thalassemia gene and a downstream primer set 1 for calculating a plurality of SNP sites of a fetal free DNA ratio;
  • upstream specific primer set 2 and downstream specific primer set 2 respectively, for specifically amplifying the above upstream specific amplification product 1 and downstream specific amplification product 1 respectively to obtain upstream specific expansion Product 2 and downstream specific amplification product 2,
  • the upstream specific primer set 2 includes an upstream primer set 2 for amplifying the ⁇ -thalassemia gene and an upstream primer set 2 for calculating a plurality of SNP sites of a fetal free DNA ratio;
  • the above-mentioned downstream specific primer set 2 comprising a downstream primer set 2 for amplifying the alpha thalassemia gene and a downstream primer set 2 for calculating a plurality of SNP sites of fetal free DNA ratio;
  • the upstream specific primer set 2 is closer to the corresponding amplification target site than the upstream specific primer set 1, and the downstream specific primer set 2 is closer to the corresponding amplification than the downstream specific primer set 1 Target site; the upstream specific primer set 2 and the 5' end of the downstream specific primer set 2 described above contain a linker sequence for a high throughput sequencing library.
  • the 5' end of the upstream specific primer set 1 and the downstream specific primer set 1 described above are provided with a modification for isolating the amplification product, preferably with a biotin modification.
  • the 3' end of the primer for the specific target site in the upstream specific primer set 1 and the 5' end of the primer corresponding to the target site in the upstream specific primer set 2 are 10-15 bases Coincident; the 3' end of the primer for the specific target site in the downstream specific primer set 1 and 10-15 bases from the 5' end of the primer for the target site in the downstream specific primer set 2 described above Coincident.
  • upstream primer set 1 sequence for amplifying the ⁇ -thalassemia gene is as shown in SEQ ID NOs: 1-7;
  • the upstream primer set 1 sequence of the above plurality of SNP sites for calculating the ratio of fetal free DNA is as shown in SEQ ID NOS: 8-17;
  • the sequence of the downstream primer set 1 for amplifying the alpha thalassemia gene is shown in SEQ ID NOs: 18-23;
  • the above upstream primer set 2 sequence for amplifying the alpha thalassemia gene is shown in SEQ ID NOs: 34-40;
  • the upstream primer set 2 sequence of the above plurality of SNP sites for calculating the ratio of fetal free DNA is shown in SEQ ID NOs: 41-50;
  • the sequence of the downstream primer set 2 for amplifying the alpha thalassemia gene is shown in SEQ ID NOs: 51-56;
  • the sequence of the downstream primer set 2 of the above plurality of SNP sites for calculating the ratio of fetal free DNA is shown in SEQ ID NOs: 57-66.
  • kit further includes:
  • the method and kit of the invention can construct a library for detection of alpha thalassemia gene mutation by using maternal free plasma DNA as a raw material, and realize detection of alpha thalassemia gene mutation by high-throughput sequencing of the library (including - ⁇ SEA , - ⁇ 3.7 , - ⁇ 4.2 , ⁇ CS , ⁇ QS , ⁇ WS mutation detection).
  • the key is that in the library construction, a specific linker is ligated to the free DNA fragment of the maternal peripheral blood, and then the pre-amplification product of the linker ligation product is creatively divided into two parts, which are used independently for the target site (such as ⁇ -type thalassemia).
  • the upstream and downstream primers of the gene are subjected to two rounds of specific amplification, which can highly enrich the target site and significantly increase the specificity of primer amplification.
  • the upstream primer set and the downstream primer set for calculating a plurality of SNP sites of the fetal free DNA ratio are respectively used for amplification.
  • different primers are also used for the SNP site for two rounds of specificity.
  • Sexual amplification can efficiently and accurately calculate the fetal DNA content.
  • the fetal DNA content can be calculated based on the sequencing read length and the thalassemia-related mutations can be classified to effectively distinguish the mutations of the embryonic gene.
  • the inventors have designed highly efficient primers specifically for thalassemia genes, as well as primers for calculating multiple SNP sites of fetal DNA ratio.
  • Using the library construction method of the present invention it is preferred to accurately and efficiently detect alpha-thalassemia gene mutations (including - ⁇ SEA , - ⁇ 3.7 , - ⁇ 4.2 , ⁇ ) using the primer sequence set provided by the present invention.
  • CS , ⁇ QS , ⁇ WS mutation detection the results are consistent with the amniocentesis test classification, but the safety, non-invasive and high efficiency is significantly better than amniocentesis.
  • FIG. 1 is a method for constructing a non-invasive prenatal fetal alpha thalassemia gene mutation detection library and a mutation detecting method according to an embodiment of the present invention; Schematic diagram of the process;
  • FIG. 2 is a schematic diagram showing the relative positional relationship between the upstream and downstream specific primers, the universal primers and the template in the embodiment of the present invention
  • M represents Marker
  • A1-A6 represents 6 An exemplary sample.
  • a method for constructing a non-invasive prenatal fetal alpha thalassemia gene mutation detection library includes the steps of:
  • S1 A linker sequence with a specific tag sequence and an optional sample tag sequence is ligated to the parental peripheral blood free DNA.
  • the parental peripheral blood free DNA that is, the peripheral blood free DNA of pregnant women, including maternal blood free DNA and fetal-derived free DNA.
  • the linker sequence carries a specific tag sequence, which can be a random sequence of n (eg, 8 or 10) bases in length, such that each linker sequence is different from the other linker sequences, that is, the linker sequence is
  • n eg, 8 or 10
  • the free DNA fragments of each parental blood bank have a specific linker sequence, which facilitates the differentiation of different maternal peripheral blood free DNA fragments in the sequence information analysis after subsequent sequencing.
  • sample pooling of different sources eg, peripheral blood free DNA from different maternal sources
  • linker sequence can also With a sample tag sequence (index), it is used to distinguish samples from different sources.
  • S2 Pre-library amplification of the ligation product of the previous step using a pre-library amplification primer sequence, wherein the pre-library amplification primer sequence is complementary to the linker sequence.
  • Pre-library amplification is performed to increase the amount of linker ligation product, as some of the molecules in the linker ligation product may have a lower copy number and are specifically amplified in subsequent partitions. At these times, these molecules with low copy number may not be efficiently amplified, and pre-amplification increases the amount of linker ligation products, so that molecules of various copy numbers can be efficiently amplified.
  • the pre-library obtained in the previous step is divided into an upstream pre-library and a downstream pre-library, and the upstream pre-library and the downstream pre-library are specifically amplified by using the upstream specific primer set 1 and the downstream specific primer set 1, respectively, respectively.
  • the pre-library is divided into an upstream pre-library and a downstream pre-library for two rounds of specific amplification, which can significantly increase the specificity of primer amplification.
  • the beneficial effects of separate independent amplification on the upstream and downstream include: on the one hand, upstream specific primers and downstream specific primers need to be closer to the amplification target site due to the second generation high-throughput sequencing read shorts (or Target region), it is difficult to effectively amplify the target fragment without separation, and independent amplification of the upstream and downstream can effectively amplify the target fragment; on the other hand, independent amplification of the upstream and downstream can retain the linker sequence at one end of the sequence. Easy for subsequent high-throughput measurements The progress of the order.
  • the amplification target site includes two types of thalassemia gene and a plurality of SNP sites for calculating fetal DNA ratio, and correspondingly, upstream specific primer set 1 and downstream specific
  • the primer set 1 also includes two primers respectively, that is, the upstream specific primer set 1 includes: an upstream primer set 1 for amplifying the ⁇ -thalassemia gene and a plurality of SNP sites for calculating a fetal free DNA ratio.
  • the upstream primer set 1; the downstream specific primer set 1 includes a downstream primer set 1 for amplifying the ⁇ -thalassemia gene and a downstream primer set 1 for calculating a plurality of SNP sites of the fetal free DNA ratio.
  • USP1 upstream primer set 1
  • DSP1 downstream primer set 1
  • Relative positional relationship It can be seen that USP1 and DSP1 are located at the left and right sides of the amplification target site, or the detection point (ie, the ⁇ -type thalassemia gene mutation). Generally, the 3' end of USP1 and DSP1 are closer to the detection point.
  • the second-generation high-throughput sequencing reads shorter, such as about 100bp, if the USP1 and DSP1 3' end distance detection point In the far distance, it may be that the second generation high-throughput sequencing cannot effectively detect the detection points.
  • the relative positional relationship of the upstream and downstream specific primers to the template binding of the plurality of SNP sites for calculating the fetal free DNA ratio is the same as that of the schematic diagram of FIG.
  • the 5' end of the upstream specific primer set 1 and the downstream specific primer set 1 may be provided with a modification for isolating the amplification product, preferably with Biotin modification, biotin can bind to avidin, so upstream specific amplification product 1 and downstream specific amplification product 1 can be separated from the reaction system by biotin-avidin affinity binding.
  • the specific primers used for the second round of specific primer amplification also include upstream specific primers and downstream specific primers.
  • the upstream specific primer set 2 used for the second round of specific primer amplification comprises: an upstream primer set 2 for amplifying the alpha thalassemia gene 2 And an upstream primer set 2 for calculating a plurality of SNP sites of the fetal free DNA ratio;
  • the downstream specific primer set 2 includes: a downstream primer set 2 for amplifying the ⁇ -thalassemia gene and for calculating a fetal free DNA ratio The downstream primer set 2 of multiple SNP sites.
  • the upstream primer set 2 (USP2) for amplifying the ⁇ -thalassemia gene and the downstream primer set 2 (DSP2) for amplifying the ⁇ -thalassemia gene in combination with the template in the embodiment of the present invention.
  • USP2 is closer to the alpha-type thalassemia gene mutation site than USP1
  • DSP2 is closer to the alpha-type thalassemia gene mutation site than DSP1.
  • the relative positional relationship between the upstream and downstream primer set 2 and the template binding of the plurality of SNP sites for calculating the fetal free DNA ratio is the same as that of the schematic diagram of FIG.
  • the upstream specific primer set 2 is closer to the corresponding amplification target site than the upstream specific primer set 1
  • the downstream specific primer set 2 is closer to the corresponding amplification target site than the downstream specific primer set 1.
  • the primer for a specific target site in the upstream specific primer set 1 for example, USP1 for the alpha-type thalassemia gene
  • the upstream specific primer set 2 for the target site Primers (eg, USP2 for the alpha-type thalassemia gene) have a 10-15 base overlap at the 5' end; similarly, primers for a specific target site in the downstream specific primer set 1 (eg, for alpha-type thalassemia)
  • the 3' end of the DSP1) of the gene has a 10-15 base overlap with the 5' end of the downstream specific primer set 2 for the target site (for example, DSP2 for the alpha-type thalassemia gene).
  • 3' end and “5' end” refer to a sequence near the 3' end and the 5' end, respectively. This coincidence is mainly considering that USP1 and DSP1 are close to the original detection point (for example, ⁇ -type thalassemia gene mutation site), and the superposition of bases can fully utilize the binding space while further improving the amplification specificity.
  • upstream specific primer set 2 and downstream specific primer set 2 may contain a linker sequence for a high throughput sequencing library. Specifically, which high-throughput sequencing platform is used, the corresponding linker sequence of the platform is used.
  • the universal primer is capable of binding to the linker sequence and is amplified by universal primers to obtain a library for sequencing on the machine. Sequencing on the machine is achieved by high-throughput sequencing.
  • the high throughput sequencing technology can be selected from the group consisting of Illumina/Genome Analyzer/Hiseq/Miseq/NEXTseq CN500, Applied Biosystems SOLID, and life Technologies/Ion Torrent. Specifically, which high-throughput sequencing technology is used, universal primers also use primers corresponding to the high-throughput sequencing technology.
  • the upstream primer set 1 sequence for amplifying the alpha-type thalassemia gene is set forth in SEQ ID NOs: 1-7; upstream of a plurality of SNP sites for calculating fetal free DNA ratio Primer set 1 sequences are set forth in SEQ ID NOS: 8-17; downstream primer set 1 sequences for amplifying alpha-type thalassemia gene are set forth in SEQ ID NOS: 18-23; used to calculate the ratio of fetal free DNA
  • the sequence of the downstream primer set 1 of one SNP site is shown in SEQ ID NOs: 24-33; the sequence of the upstream primer set 2 used to amplify the alpha thalassemia gene is shown in SEQ ID NOs: 34-40;
  • the upstream primer set 2 sequence of the plurality of SNP sites of the fetal free DNA ratio is shown in SEQ ID NOs: 41-50;
  • the downstream primer set 2 sequence for amplifying the alpha thalassemia gene is SEQ ID NO: 51-56
  • the embodiment of the invention further provides a non-invasive prenatal fetal alpha thalassemia gene mutation detecting method, comprising performing high-throughput sequencing of the library constructed in the embodiment of the invention to obtain a sequencing read read; then, according to the unique position of the site Specific tag sequence The total depth and depth of the allele of the mutation, the fetal DNA content was calculated and the thalassemia-related mutations were typed to analyze the alpha-thalassemia gene mutation.
  • the embodiment of the invention further provides a non-invasive prenatal fetal alpha thalassemia gene mutation detection library construction kit, wherein the above library is used for detecting non-invasive prenatal fetal alpha thalassemia gene mutation by high-throughput sequencing, the above kit include:
  • upstream specific primer set 2 comprises an upstream primer set 2 for amplifying the alpha-thalassemia gene and an upstream of a plurality of SNP sites for calculating the fetal free DNA ratio Primer set 2; downstream specific primer set 2 includes a downstream primer set 2 for amplifying the alpha-thalassemia gene and a downstream primer set 2 for calculating a plurality of SNP sites of the fetal free DNA ratio.
  • upstream specific primer set 2 is closer to the corresponding amplification target site than the upstream specific primer set 1, and the downstream specific primer set 2 is closer to the corresponding amplification target site than the downstream specific primer set 1;
  • the 5' end of upstream specific primer set 2 and downstream specific primer set 2 contains a linker sequence for a high throughput sequencing library.
  • the upstream specific primer set 1 and the downstream specific primer set 1 have a modification for the isolation of the amplified product, preferably with a biotin modification, to separate upstream specific expansion from the system.
  • Product 1 and downstream specific amplification product 1 are added.
  • the 3' end of the primer for the specific target site in the upstream specific primer set 1 and the 10' end of the primer for the target site in the upstream specific primer set 2 are 10-15 Coincident of bases; the 3' end of the primer for the specific target site in the downstream specific primer set 1 and the 10' end of the primer of the downstream specific primer set 2 for the target site are 10-15 bases Coincident.
  • the upstream primer set 1 sequence for amplifying the alpha-type thalassemia gene is set forth in SEQ ID NOs: 1-7; upstream of a plurality of SNP sites for calculating fetal free DNA ratio Primer set 1 sequences are set forth in SEQ ID NOS: 8-17; downstream primer set 1 sequences for amplifying alpha-type thalassemia gene are set forth in SEQ ID NOS: 18-23; used to calculate the ratio of fetal free DNA
  • the sequence of the downstream primer set 1 of one SNP site is shown in SEQ ID NOs: 24-33; the sequence of the upstream primer set 2 used to amplify the alpha thalassemia gene is shown in SEQ ID NOs: 34-40;
  • the upstream primer set 2 sequence of multiple SNP sites of fetal free DNA ratio is shown in SEQ ID NOs: 41-50; used to amplify alpha-type thalassemia
  • the sequence of the downstream primer set 2 is shown in SEQ ID NOS: 51-56; the
  • the kit further comprises:
  • R3 linker sequence carrying a specific tag sequence and an optional sample tag sequence for ligation of maternal peripheral blood free DNA to obtain a ligation product
  • R4 a pre-library amplification primer sequence which is complementary to the above-described linker sequence for pre-library amplification of the above-described ligation product
  • R5 a universal primer for amplifying the mixed product after mixing the upstream specific amplification product 2 and the downstream specific amplification product 2 described above to obtain a library which can be sequenced on the machine.
  • the linker sequence is set forth in SEQ ID NOs: 67-68; the pre-library amplification primer sequences are set forth in SEQ ID NOs: 69-70; and the universal primers are set forth in SEQ ID NO: 71-72. Show.
  • the upstream primer and the downstream primer are located on the left and right sides of the detection point, respectively.
  • the 3' end of the ipsilateral specific primer 1 and the 5' end of the specific primer 2 have a 10-15 base overlap.
  • Specific primer 1 has a biotin modification at the 5' end.
  • the 5' end of specific primer 2 contains a linker sequence for a high throughput sequencing library. Specifically, the primer sequences are shown in Table 1.
  • ADT-R pGATCGGAAGAGC (SEQ ID NO: 68).
  • NNNNNNNN is a specific tag sequence
  • ATTAAGG is a sample tag sequence (index)
  • the above linker sequence needs to be annealed into a double strand.
  • pre-lib-primer-F CAAGCAGAAGACGGCATACGA (SEQ ID NO: 69);
  • pre-lib-primer-R GCTCTTCCGATCT (SEQ ID NO: 70).
  • Seq-lib-primer-F CAAGCAGAAGACGGCATACGA (SEQ ID NO: 71);
  • reaction 1 Free DNA end repair, and the reaction system (Reaction 1) was set as shown in Table 2.
  • reaction 2 The DNA fragment is ligated to the linker sequence carrying the specific tag sequence and the sample tag sequence, and the reaction system (Reaction 2) is shown in Table 3.
  • thermocycler 20 ° C, 15 min; 65 ° C, 10 min; 4 ° C preservation.
  • the final reaction product was purified by using 50 ⁇ L of XP beads, and quantification by Qubit, and 5 ⁇ L of the reaction product was detected by 2% gel electrophoresis. The results are shown in Fig. 3a, indicating that the pre-library was successfully constructed. Two aliquots of the pre-library were taken and named as the upstream pre-library and the downstream pre-library, respectively, and each 100 ng was subjected to the next amplification.
  • Each sample pre-library is an upstream pre-library and a downstream pre-library, respectively using the corresponding upstream and downstream specific primer 1 and specific primer 2, respectively, until the amplification of the upstream and downstream primers of each sample is combined when using universal primer amplification. And then use universal primers for amplification.
  • the PCR reaction procedure was as follows: 95 ° C, 10 min, 1 cycle; (95 ° C, 30 s, 62 ° C, 30 s, 72 ° C, 1 min) 20 cycles; 72 ° C, 7 min, 1 cycle; 4 ° C storage.
  • reaction product 5 ⁇ L was subjected to 2% agarose gel electrophoresis, and the remaining product was recovered by 1.2X XP, eluted with 24 ⁇ L of ultrapure water or an eluent, and the eluate was subjected to further amplification.
  • the PCR reaction procedure was as follows: 95 ° C, 10 min, 1 cycle; (95 ° C, 30 s, 62 ° C, 30 s, 72 ° C, 1 min) 15 cycles; 72 ° C, 7 min, 1 cycle; 4 ° C preservation.
  • the PCR reaction procedure was as follows: 98 ° C, 45 s, 1 cycle; (98 ° C, 15 s, 60 ° C, 30 s, 72 ° C, 30 s) 10 cycles; 72 ° C, 1 min, 1 cycle; 4 ° C preservation.
  • Illumina NEXTseq CN500 was subjected to 75PE sequencing.
  • the molecular tag set sequences were re-aligned to the reference genome (BWA MEM), the sequences surrounding Indel were re-aligned using GATK software, SNP and small indels were detected using samtools software, and alpha1 ( ⁇ 1) and alpha2 were performed using CNVPanelizer R package. ( ⁇ 2) deletion detection. Calculate the embryonic DNA content based on the total depth of the unique molecular tag set sequence of the locus and the depth of the associated SNP locus allele, and genotype the fetal thalassemia-related point mutation, INDEL, for alpha1 And alpha2 type deletion, mixed Gaussian model for fetal genotyping.
  • non-invasive prenatal thalassemia gene detection was performed on 8 pregnant women with thalassemia carriers, and the results are shown in Table 8 below.
  • results show that the non-invasive prenatal detection and the amniocentesis detection of the ⁇ -thalassemia gene mutation are consistent in the embodiment of the present invention, indicating that the method of the present invention can accurately and efficiently detect the alpha-thalassemia gene mutation, and the result and the amniocentesis test score.
  • the type is consistent, but it is significantly better than amniocentesis in terms of safety, non-invasiveness and high efficiency.

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Abstract

L'invention concerne un procédé de construction, un procédé de détection et un kit pour une bibliothèque de détection de mutation de gène de thalassémie alpha fœtale prénatale non invasive. Dans le procédé de construction de la bibliothèque, un lieur spécifique est ligaturé à un fragment d'ADN libre dans le sang périphérique maternel, et le produit de pré-amplification du produit de ligature lieur est ensuite divisé en deux parties, et deux cycles d'amplification spécifique sont respectivement réalisés indépendamment à l'aide d'amorces ascendantes et descendantes pour un site cible, et le site cible peut être enrichi avec une spécificité élevée, et la spécificité d'amplification des amorces peut être considérablement améliorée. De plus, deux cycles d'amplification spécifique sont respectivement réalisés à l'aide d'ensembles d'amorces ascendantes et descendantes pour de multiples sites SNP pour calculer la proportion d'ADN fœtal libre, et la proportion d'ADN fœtal peut être calculée de manière efficace et précise. Le séquençage de la bibliothèque peut détecter avec précision et efficacité la mutation du gène de thalassémie alpha, et les résultats de celle-ci sont cohérents avec ceux du génotypage de détection par amniocentèse ; cependant, en termes de sécurité, de non-invasivité et d'efficacité élevée, le procédé est significativement meilleur que la détection par amniocentèse.
PCT/CN2017/113231 2017-01-19 2017-11-28 Procédé de construction, procédé de détection et kit pour une bibliothèque de détection de mutation de gène de thalassémie alpha fœtale prénatale non invasive WO2018133546A1 (fr)

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CN107099609A (zh) * 2017-06-19 2017-08-29 东莞市妇幼保健院 一种微滴式数字pcr检测低比例痕量目标基因的遗传分析方法
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CN113012759B (zh) * 2020-12-09 2022-08-12 人和未来生物科技（长沙）有限公司 一种基于X染色体的男胎cffDNA含量计算方法
CN112831555B (zh) * 2021-02-01 2022-12-20 人和未来生物科技（长沙）有限公司 一种检测地中海贫血基因的参考品及其制备方法与应用

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