WO2019132582A1 - Method for analyzing str of human subject by using multiplex system, and analysis kit using same - Google Patents

Method for analyzing str of human subject by using multiplex system, and analysis kit using same Download PDF

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WO2019132582A1
WO2019132582A1 PCT/KR2018/016847 KR2018016847W WO2019132582A1 WO 2019132582 A1 WO2019132582 A1 WO 2019132582A1 KR 2018016847 W KR2018016847 W KR 2018016847W WO 2019132582 A1 WO2019132582 A1 WO 2019132582A1
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human
gene
primer
str
multiplex
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Korean (ko)
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정영주
정지나
이재원
김광중
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주식회사 엔젠바이오
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/00—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
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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    • C12Q2537/00—Reactions characterised by the reaction format or use of a specific feature
    • C12Q2537/10—Reactions characterised by the reaction format or use of a specific feature the purpose or use of
    • C12Q2537/143—Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
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    • 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
    • C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
    • C12Q2565/10—Detection mode being characterised by the assay principle
    • C12Q2565/125—Electrophoretic separation
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention relates to a method for analyzing a STR of a human subject using dual multiplex gene amplification, and more particularly, to a method for analyzing STR of a human subject using 24 dual STR loci (DNA polymerase chain reaction) using dual multiplex polymerase chain reaction short tandem repeat loci) is amplified and analyzed to give rapid and high-accuracy gene identification, and a kit using the same.
  • dual STR loci DNA polymerase chain reaction
  • dual multiplex polymerase chain reaction short tandem repeat loci is amplified and analyzed to give rapid and high-accuracy gene identification, and a kit using the same.
  • PCR Polymerase Chain Reaction
  • Short Tandem Repeat sequences known to be variously present in introns that do not contain any genetic information such as traits in human DNA are being used globally as markers of gene emotion, Has an STR site with this polymorphism.
  • the STR region has a repeat structure of 2 to 7 nucleotides. As the number of short repeats in a specific locus differs, the length of DNA in this locus differs for each allele, Alleles. Thus, individual identification is possible according to the STR repeat number (allele) of these specific loci.
  • sex chromosomal amelogenin loci can be used to identify human sex.
  • Amelogenin locus is identified as HUMAMELX when identifying the locus on the Y chromosome present in the male DNA in the gene bank (GenBank) and as HUMAMELX when identifying the locus on the X chromosome present in the female DNA.
  • a multiplex PCR system is one in which a primer of several loci is inserted into a single PCR reaction and an experimental reaction is performed in a single tube.
  • taq Polymerase PCR reaction enzyme
  • other reagents as well as minimizing the operation of the genotype capillary automatic analyzer, thereby reducing the cost.
  • Gene detection has the advantage of being able to save considerably on budget considering that expensive commercial reagents are consumed. In this way, researches have been actively conducted to establish a multiplex PCR system suitable for the local reality in advanced countries such as the United Kingdom and the United States leading to gene identification due to the utility value of the multiplex PCR system.
  • triplex PCR kit II
  • Promega Corporation Promega Corporation
  • Triplex PCR kit II
  • Promega Corporation Promega Corporation
  • studies and development centered on triplex PCR systems have been actively conducted in the early days (Kimpton et al., 1993 Urquhart et al., 1994).
  • the development of the Quadruplex system (Sprecher et al., 1996; Lins et al., 1996; Robertson JM et al., 1995), which simultaneously analyzes four genetic loci, (Kimpton et al., 1996; Yest et al., 1997) have also been performed.
  • Thermofisher has developed and marketed D3S1358, VWA, D16S539, CSF1PO, TPOX, Yindel, Amelogenin, D8S1179, D21S11, D18S51, DYS391, D2S441, D19S433, TH01, FGA, D22S1045, D5S818, D13S317, D7S820, SE33, D10S1248, D1S1656, Simultaneous amplification and four-color detection of 24 genetic loci including D2S1338 have been developed (Globalfiler).
  • Promega was able to perform simultaneous amplification of 16 genetic loci including D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, Amelogenin, VWA, D8S1179, TPOX, 4-color detection method was developed (Powerplex 16).
  • the somatic chromosome STR amplification method widely used in Korea is also a commercial kit manufactured and sold by a foreign company. This is because the STR markers developed in the US and UK, which led to the development of STR analysis method by PCR in the late 1990s, were acquired by the companies in the respective countries and commercialized as kits. In particular, as the criminal DNA database became globally universal, it was recommended that all countries use common STR markers for the exchange of DNA information between countries, so that later generations will use commercial kits that include a broad range of STR markers .
  • the above-mentioned commercialization kit has a merit that it is convenient, but it has a disadvantage that it relies on foreign company's technology.
  • the companies that produce these kits are, as we have seen, Applied Biosystems and Promega, which are a major company, with over 10 commercial kits being produced and sold.
  • the kits for these two companies, which have dominated the global market, are very expensive and represent a large part of the cost of genetic testing.
  • KR 10-1672240 discloses an STR analysis method on a human object chromosome and an assay kit using the same
  • KR 10-1667526 discloses a method of analyzing an expanded human autologous chromosome STR using a next-generation sequencing method.
  • KR 10-0277289 discloses a Quadruplex PCA system composed of Esterella loci with differentiating power in a Korean population and a gene detection method using the same
  • KR 10-1008828 discloses 16 short repeats Unit genetic loci and sex chromosomal genetic loci, and a method for gene detection using the same, discloses a method of gene detection using a multiplex Y gene chromosome gene amplification system in KR 10-1414827
  • KR 10-1533792 discloses an autosomal analysis technique for NGS-based human subjects
  • KR 10-1457983 discloses a method for analyzing human autosomes using a multiplex gene amplification method.
  • KR 10-0443569 a multiplex amplification polymerase Discloses a method of identifying a strand by discrimination of a strand and a strand discrimination method by optimizing a chain reaction condition.
  • KR 10-1341943 discloses an STR detection kit and a STR detection method using the same, and in KR 10-1716108, Discloses a gene detection method using preamplification, KR 10-1198096 discloses a specific primer for identification or paternity identification and its use, and KR 10-2010-0081968 discloses a method of identifying a human X chromosome short chain repeat Discloses a kit and method for simultaneous amplification of a nucleotide sequence, and KR 10-2009-0111407 discloses a kit for the simultaneous amplification of a human chromosome short chain repeating base sequence US 9,090,943 discloses an analysis method of genetic loci of SE33 (ACTBP2) using PCR, and US 9,797,841 discloses a STR genetic library
  • the present inventors have made intensive efforts to develop a method for analyzing a human subject having high sensitivity and accuracy.
  • a STR genome locus of 16 residues and a STR genome locus of 9 loci by a dual multiplex system, It is possible to perform the analysis with the conventional machine without using the fluorescent dye and to detect the genetic information of the sample with high sensitivity and accuracy, and the present invention has been completed.
  • Another object of the present invention is to provide a multiplex gene amplification kit for STR (short tandem repeat) analysis on a human subject chromosome comprising a set of primers capable of amplifying 24 genetic loci.
  • STR short tandem repeat
  • the present invention also provides an assay method for gene detection of a human object using multiplex gene amplification.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising (a) D8S1179, CSF1PO (Human c-fms protooncogene for CSF-1 receptor gene), D3S1358, D2S1338, Amelogenin, von Willebrand factor A, TH01, D18S51, D5S818, A first primer set comprising primers capable of amplifying each of the genetic loci comprising D21S11, human thyroid peroxidase gene (TPOX), human fibrinogen alpha chain (FGA), D13S317, D7S820, D16S539 and D19S433; And a second primer set comprising primers capable of amplifying each of the genes including amelogenin, SE33, D1S1656, Penta D, D22S1045, D10S1248, D12S391, D2S441 and Penta E and a human primate set ; And
  • step (b) determining an allele of the genetic locus using the multiplex amplification product of step (a).
  • the present invention also relates to the use of a polynucleotide selected from the group consisting of D8S1179, CSF1PO (Human c-fms protooncogene for CSF-1 receptor gene), D3S1358, D2S1338, Amelogenin, von Willebrand factor A, TH01, D18S51, D5S818, D21S11, (Human thyroid peroxidase gene), a human fibrinogen alpha chain (FGA), D13S317, D7S820, D16S539 and D19S433; And a second primer set comprising a primer capable of amplifying each of the genes including amelogenin, SE33, D1S1656, Penta D, D22S1045, D10S1248, D12S391, D2S441 and Penta E, A multiplex gene amplification kit for analyzing short tandem repeat (STR)
  • STR short tandem repeat
  • FIG. 1 is a schematic view showing positions of a genetic locus analyzed in a dual multiplex system according to the present invention for each fluorescent marker.
  • FIG. 2 shows a result of a sample analysis using a first primer set in a dual multiplex system according to the present invention.
  • FIG 3 shows a result of a sample analysis using a second primer set in a dual multiplex system according to the present invention.
  • the term 'STR' in the present invention refers to a tandem repeat sequence known to be widely distributed within an intron that does not contain any genetic information such as a trait in the human genome, ), which has been widely used worldwide.
  • Many genetic loci in the human genome contain the STR region of the polymorphism.
  • the STR locus consists of a short repeat sequence element, 2 to 7 base pairs in length, which is estimated to have 2 million trimer and tetramer STRs once every 15 kb in the human genome. As the number of short repeating units in a particular locus changes, the DNA length at that locus will vary with each allele and each individual.
  • STR loci can be amplified by Polymerase Chain Reaction (PCR) using specific primer sequences identified on the side of this repeat sequence.
  • " amplification " in the present invention means a reaction for amplifying a nucleic acid molecule.
  • a variety of amplification reactions have been reported in the art, including polymerase chain reaction (PCR) (US Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), reverse transcription-polymerase chain reaction (RT- , The methods of WO 89/06700 and EP 329,822, ligase chain reaction (LCR, WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification MA, WO 88/10315), self sustained sequence replication (WO 90/06995), selective amplification of target polynucleotide sequences (US Pat. No.
  • PCR consensus sequence PCR was performed using the primer polymerase chain reaction (CP-PCR, US Pat. No. 4,437,975), arbitrary primed polymerase chain reaction (AP-PCR, 5,413,909 and 5,861,245), nucleic acid sequence based amplification (NASBA, U.S. Pat. Nos. 5,130,238, 5,409,818, 5,554,517 and 6,063,603), strand displacement but are not limited to, amplification and loop-mediated isothermal amplification (LAMP).
  • CP-PCR primer polymerase chain reaction
  • AP-PCR arbitrary primed polymerase chain reaction
  • NASBA nucleic acid sequence based amplification
  • LAMP loop-mediated isothermal amplification
  • PCR is the most well-known nucleic acid amplification method, and many variations and applications thereof have been developed. For example, touchdown PCR, hot start PCR, nested PCR and booster PCR have been developed by modifying traditional PCR procedures to enhance the specificity or sensitivity of PCR.
  • real-time PCR, differential display PCR, D-PCR, rapid amplification of cDNA ends (RACE), DL-PCR (PC), inverse polymerase chain reaction inverse polymerase chain reaction (IPCR), vectorette PCR, and thermal asymmetric interlaced PCR (TAIL-PCR) have been developed for specific applications.
  • RACE rapid amplification of cDNA ends
  • PC DL-PCR
  • IPCR inverse polymerase chain reaction inverse polymerase chain reaction
  • TAIL-PCR thermal asymmetric interlaced PCR
  • the multiplex amplification is a multiplex PCR (Polymerase Chain Reaction) amplification.
  • the multiplex PCR amplification has an annealing temperature condition of 57-61 ° C.
  • the multiplex PCR amplification is performed at a temperature of 58-60 ° C Temperature conditions, and according to certain embodiments of the present invention, the multiplex PCR amplification has an annealing temperature condition of 58.5-59.5 [deg.] C.
  • the multiplex PCR amplification requires a reasonable number of cycles to perform PCR. According to one embodiment of the invention, the multiplex PCR amplification is performed in 27-29 cycles. When the multiplex PCR amplification of the present invention was carried out in 26 cycles or less, peaks of 500 RFU or less were formed and peaks of 2,000 RFU or more were formed in 30 cycles, but noise was increased and incomplete A insertion occurred.
  • &quot dual multiplex system " in the present invention means that a nucleic acid sample obtained from one sample is simultaneously subjected to multiplex system two or more times using each independent primer set.
  • primer refers to a primer that is capable of hybridizing under appropriate conditions (i.e., a single nucleotide which can act as a starting point for template-directed DNA synthesis under four different nucleoside triphosphates and polymerase,
  • the appropriate length of the primer is typically 15 to 30 nucleotides, depending on various factors, for example, temperature and use of the primer.
  • the short primer forms a sufficiently stable hybridization complex with the template.
  • a set of primers includes a nucleotide sequence It is not necessary to have a perfectly complementary sequence, and it is considered sufficient if it has sufficient complementarity within a range that it can hybridize to this sequence to give primer action.
  • the design of such a primer refers to the nucleotide sequence of a polynucleotide to be a template Can be easily carried out by a person skilled in the art, for example, by using a program for primer design (for example, PRIMER 3, VectorNTI program).
  • a human object DNA sample is treated with D8S1179, CSF1PO (Human c-fms protooncogene for CSF-1 receptor gene), D3S1358, D2S1338, Amelogenin, vWA (von Willebrand factor A)
  • a first primer set comprising primers capable of amplifying respective genetic loci composed of TH01, D18S51, D5S818, D21S11, human thyroid peroxidase gene (TPOX), human fibrinogen alpha chain (FGA), D13S317, D7S820, D16S539 and D19S433;
  • a second primer set containing primers capable of amplifying each of the genes composed of amelogenin, SE33, D1S1656, Penta D, D22S1045, D10S1248, D12S391, D2S441 and Penta E, As a result of determining allele genotypes of each genetic locus, it was confirmed that genes
  • the present invention provides, in one aspect, (a) a compound selected from the group consisting of D8S1179, CSF1PO (human c-fms protooncogene for CSF-1 receptor gene), D3S1358, D2S1338, Amelogenin, vWA, A first primer set comprising primers capable of amplifying each of the genetic loci comprising D18S51, D5S818, D21S11, Human thyroid peroxidase gene (TPOX), Human fibrinogen alpha chain (FGA), D13S317, D7S820, D16S539 and D19S433; And a second primer set comprising primers capable of amplifying each of the genes including amelogenin, SE33, D1S1656, Penta D, D22S1045, D10S1248, D12S391, D2S441 and Penta E and a human primate set ; And (b) determining an allelic genotype of the genetic locus using the multiplex
  • the present invention is directed to a method for modulating the expression of a protein selected from the group consisting of (a) D8S1179, CSF1PO (Human c-fms protooncogene for CSF-1 receptor gene), D3S1358, D2S1338, Amelogenin, vWA , Primers capable of amplifying each of the genetic loci including TH01, D18S51, D5S818, D21S11, human thyroid peroxidase gene (TPOX), human fibrinogen alpha chain (FGA), D13S317, D7S820, D16S539 and D19S433 set; And a second primer set comprising primers capable of amplifying each of the genes including amelogenin, SE33, D1S1656, Penta D, D22S1045, D10S1248, D12S391, D2S441 and Penta E and a human primate set ; And (b) determining an allele of the genetic locus
  • the primer may be characterized by being capable of amplifying STR strands, maintaining the GC ratio in the range of 40 to 60%, and complementary to the gene sequence having no repeating sequence of nucleotides.
  • the first primer set may be characterized by being represented by the nucleotide sequences of SEQ ID NOS: 1-32.
  • the second primer set may be a primer represented by the nucleotide sequence of SEQ ID NOS: 33 to 50.
  • the amplification product of step (a) may be 80 to 400 bp.
  • step (b) is performed by comparing the size of the allele amplified in the multiplex amplification product with a size standard, and the size standard is a DNA marker or a genetic locus- Specific allele gene ladder.
  • the DNA sample may be a DNA sample isolated from a tissue selected from the group consisting of blood, semen, vaginal cells, hair, saliva, urine, oral cells, placental cells or fetal cells, .
  • the biological sample may be selected from the group consisting of blood, hair, saliva, urine, oral cells, and mixtures thereof.
  • gene detection of a human object means determining the genotype of an object by determining an allele genotype and comparing it with a control. For example, when gene detection of DNA in a blood sample found at a crime scene is performed by the method of the present invention, it is possible to determine whether the determined allelotype exists in the criminal interface, to identify the suspect, , It means to determine the allelic genotype of the sample object, and to compare the allelic genotype of the father or the progeny with the control group to confirm the parental status.
  • the first primer set and the second primer set may be labeled with a fluorescent dye.
  • the primer set represented by the nucleotide sequences of SEQ ID NOS: 1 to 8 and SEQ ID NOS: 33 to 34 is labeled with the first fluorescent dye
  • a primer set represented by the nucleotide sequences of SEQ ID NOS: 17 to 24 and SEQ ID NOS: 41 to 46 are labeled with a third fluorescent dye
  • the primer set represented by the nucleotide sequence of 47 to 50 can be characterized as being labeled with a fourth fluorescent dye.
  • a fluorescent dye is labeled at the 5 'end of one primer of a pair of primers complementarily binding to the genetic locus.
  • the first to fourth fluorescent dyes are labels for constituting three to four genetic loci in a lane so that they can be detected through capillary electrophoresis.
  • the fluorescent dye may be at least one selected from the group consisting of 6-FAM, VIC, NED, PET, ROX, HEX, dR110, dR6G, dTAMRA and dROX.
  • the primer used in the gene detection method of the present invention has an optimal blending ratio for adjusting the peak balance.
  • the primer that binds complementarily to the D8S1179 genetic locus in the first primer set has a final concentration of 1.0-5.0 [mu] M and the primer that binds complementarily to the CSF1PO genetic locus is 0.1-2.0 [mu] M
  • Primers complementarily binding to the D3S1358 genetic locus have a final concentration of 0.1-2.0 [mu] M
  • primers that bind complementarily to the D2S1338 genetic locus have a final concentration of 0.5-3.0 [mu] M
  • the Amelogenin genetic Primers that complementarily bind to the locus have a final concentration of 0.1-2.0 [mu] M
  • primers that bind complementarily to the vWA genetic locus have a final concentration of 1.0-5.0 [mu] M
  • primers that complementarily bind to the TH01 genetic locus Has a final concentration of 0.1-2.0 [mu] M
  • Electrophoresis relates to the flow of particles under the influence of the same spatially distributed electric field.
  • the types of electrophoresis include, but are not limited to affinity electrophoresis, capillary electrophoresis, and gel electrophoresis.
  • capillary electrophoresis is used to isolate the amplified allele in the multiplex amplification product of the present invention.
  • an electric field is applied to the charged ions, and each of the ions moves to the electrode having the opposite charge.
  • the movement speed depends on the average charge, size, shape, and properties of the solvent.
  • Capillary electrophoresis is a technique that utilizes the property that when ions are applied to both ends of a tube in a state where ions are present through a thin tube, the ions move in the tube with different orientations at different rates according to their properties. It is a device to separate.
  • a compound of the present invention which comprises a D8S1179, a CSF1PO (Human c-fms protooncogene for CSF-1 receptor gene), D3S1358, D2S1338, Amelogenin, vWA, TH01, D18S51, D5S818, , A human thyroid peroxidase gene (TPOX), a human fibrinogen alpha chain (FGA), D13S317, D7S820, D16S539 and D19S433; And a second primer set comprising a primer capable of amplifying each of the genes including amelogenin, SE33, D1S1656, Penta D, D22S1045, D10S1248, D12S391, D2S441 and Penta E, And more particularly to a multiplex gene amplification kit for analyzing STR (short tandem repeat)
  • the kit may further comprise a reaction buffer, a DNA polymerase, dNTP (a mixture comprising dATP, dCTP, dGTP and dTTP).
  • the DNA polymerase may be, for example, a thermostable DNA polymerase obtained from Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis or Pyrococcus furiosus (Pfu).
  • the reaction buffer is a compound added to an amplification reaction that modifies the stability, activity, and / or lifetime of one or more components of the amplification reaction by adjusting the pH of the amplification reaction.
  • buffer solutions are well known in the art, For example, Tris, Tricine, MOPS, or HEPES.
  • the kit may further comprise a DNA polymerase joiner, if desired.
  • Example 1 Obtaining a DNA sample from a sample
  • a DNA sample was obtained in the following manner (E-Prep DNA extraction solution, Viagen Biotech):
  • E-Prep Solution (Viagen Biotech)
  • 10 ⁇ l of Porteinase K (20 mg / ml, Sigma Aldrich) was added and centrifuged to collect the collected sample
  • the cells were cultured in a constant temperature water bath at 63 ° C for 20 minutes and then cultured in a constant temperature water bath at 85 ° C for 15 minutes and then centrifuged at 13,000 rpm / min for 4 minutes to recover 50-100 ⁇ l of the supernatant.
  • a DNA sample was obtained by the following method (Qiagen human genomic DNA extraction kit, Qiagen, USA):
  • ATL buffer (Qiagen, USA) was injected into the sample and incubated for 1 hour in a constant temperature water bath at 56 ° C. The whole supernatant was recovered and transferred to another tube, and then AL buffer (Qiagen, After incubating for 10 minutes in a constant-temperature water bath at 70 ° C, 200 ⁇ l of 100% ethanol was added to the column, and the solution was centrifuged at 13,000 rpm for 1 minute to discard the solution in the collection tube and 650 ⁇ l of AW1 buffer After centrifugation at 13,000 rpm for 1 minute, the solution in the collection tube was discarded and the procedure was repeated twice. The washed column was inserted into a 1.5 ml tube, and then 20 ⁇ l of tertiary distilled water was added thereto. Minute, centrifuged at 13,000 rpm for 1 minute to obtain final genomic DNA.
  • the PCR product was prepared in a volume of 20 ⁇ l based on a 2 ⁇ PCR master mix (Qiagen, USA), 10 ⁇ l of sample DNA, 2 ⁇ l of 10 ⁇ primer set, and DW buffer.
  • the minimum detection limit of DNA samples was 0.5 to 1 ng Due to the nature of the medical samples, it was confirmed that a profile corresponding to a trace amount of the sample had a sensitivity that could be detected using this kit.
  • Dual multiplex PCR reactions were performed under the following conditions.
  • Amplification products were determined using capillary electrophoresis using a 3130XL Genetic Analyzer (ABI, USA), a 3730XL Genetic Analyzer (ABI, USA) and a 3500 or 3500XL Genetic Analyzer (ABI, USA).
  • the present invention relates to a method for amplifying a large amount of fluorescently labeled sample DNA using fluorescence-labeled PCR primer set (polymerase chain reaction primer set) and then amplifying the amplified DNA with a DNA sequencer (genetic analyzer) ,
  • a base-pair marker size standard that corrects experimental errors is required to obtain the exact number of base pairs of amplified fluorescent-labeled DNA.
  • DNA allelic ladder This is called DNA allelic ladder. All STR kit requires DNA allelic ladder suitable for each kit. DNA allelic ladder is used to select homogenous STR from clinical specimen used in STR search using PCR primer set of kit, PCR amplification products were cloned into a carrier plasmid (vector plasmid) to prepare an allelic ladder library. The Allelic ladder library was PCR-amplified by PCR to match the size of each base-pair of STR, and the products were mixed to complete the concentration control to adjust each peak balance.
  • the analysis method for gene detection of a human object according to the present invention is useful for identification of individuals and identification of blood vessels because human identification can be performed with high reproducibility and accuracy in a situation where a dependency of imported products is 100% .

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Abstract

The present invention relates to a method for analyzing the STR of a human subject to be analyzed by using dual multiplex gene amplification and, more specifically, to: a method using dual multiplex polymerase chain reaction (PCR) so as to simultaneously amplify and analyze 24 short tandem repeat (STR) loci, thereby enabling rapid and highly accurate gene identification; and a kit using the same. The analysis method for gene identification of a human subject, according to the present invention, can identify genes with high reproducibility and accuracy in a situation in which human gene identification has 100% dependency on imported goods, thereby being usable in individual identification and blood relative identification.

Description

듀얼 멀티플렉스 시스템을 이용한 인간 객체의 STR 분석방법 및 이를 이용한 분석 키트STR analysis method of human object using dual multiplex system and analysis kit using it
본 발명은 듀얼 멀티플렉스 유전자 증폭을 이용한 분석대상의 인간 객체의 STR 분석 방법에 관한 것으로, 더욱 자세하게는 듀얼 멀티플렉스 중합효소연쇄반응(PCR; polymerase chain reaction)을 이용하여 24개 STR 유전좌위들(short tandem repeat loci)를 등시에 증폭하여 분석함으로써 신속하고 높은 정확도로 유전자 감식을 할 수 있는 방법 및 이를 이용한 키트에 관한 것이다.The present invention relates to a method for analyzing a STR of a human subject using dual multiplex gene amplification, and more particularly, to a method for analyzing STR of a human subject using 24 dual STR loci (DNA polymerase chain reaction) using dual multiplex polymerase chain reaction short tandem repeat loci) is amplified and analyzed to give rapid and high-accuracy gene identification, and a kit using the same.
개인식별이나 혈연관계 여부 확인을 위한 유전자감정은 중합효소연쇄반응(PCR; Polymerase Chain Reaction) 이용을 기본으로 한 방법이 본격적으로 사용되고 있으며, 이러한 원리는 범죄 사건 등에 법의학적 타이핑이나 DNA 신원 확인 정보와 같은 DNA 데이터베이스 구축하는데 활용되고 있다. 인간 DNA 내에 어떤 형질 등을 나타내는 유전 정보를 담고 있지 않은 인트론(intron) 내에 다양하게 존재하는 것으로 알려진 짧은 반복 염기 서열(Short Tandem Repeat sequence)들은 유전자 감정의 마커로서 전 세계적으로 유용하게 사용되고 있으며 많은 유전자좌가 이러한 다형성을 갖는 STR 부위를 가지고 있다. STR 부위는 2개에서 7개의 염기 서열이 반복되는 구조로 되어 있는데, 특정 좌위에서의 짧은 반복 배열 수가 다름에 따라 이 좌위에서의 DNA 길이가 각 대립 형질(allele)에 따라 다르고 개인에 따라 고유의 대립 형질 갖게 된다. 따라서 이러한 특정 유전자좌의 STR 반복수(대립 형질)에 따라 개인식별이 가능하다. 또한, 인간의 성별을 식별하기 위하여 성염색체 아멜로게닌 좌위가 사용될 수 있다. 아멜로게닌 좌위는 유전자 은행(GenBank)에서 남성 DNA에 존재하는 Y 염색체 상의 좌위를 확인하는 경우 HUMAMELY로서 여성 DNA에 존재하는 X 염색체 상의 좌위를 확인하는 경우 HUMAMELX 로서 확인된다.In order to identify individuals or identify blood relatives, a method based on PCR (Polymerase Chain Reaction) is being used in earnest. These principles are used for criminal cases, forensic typing, DNA identification information, It is being used to construct the same DNA database. Short Tandem Repeat sequences known to be variously present in introns that do not contain any genetic information such as traits in human DNA are being used globally as markers of gene emotion, Has an STR site with this polymorphism. The STR region has a repeat structure of 2 to 7 nucleotides. As the number of short repeats in a specific locus differs, the length of DNA in this locus differs for each allele, Alleles. Thus, individual identification is possible according to the STR repeat number (allele) of these specific loci. In addition, sex chromosomal amelogenin loci can be used to identify human sex. Amelogenin locus is identified as HUMAMELX when identifying the locus on the Y chromosome present in the male DNA in the gene bank (GenBank) and as HUMAMELX when identifying the locus on the X chromosome present in the female DNA.
한편, 멀티플렉스 PCR 시스템은 한번의 PCR 반응으로 여러 유전좌위들(loci)의 프라이머를 넣고 한 튜브 내에서 실험반응을 수행하는 것으로서, 멀티플렉스 PCR 시스템을 사용하게 되면 감정인력 및 분석에 사용되는 taq 폴리머라아제(PCR 반응효소) 등 각종의 시약류의 절감 효과와 함께 유전자형 모세관 자동분석기의 가동을 최소화시켜 비용을 줄일 수 있는 큰 장점이 있다. 유전자감식에는 상용화된 고가의 반응시약류들이 소모되는 것을 감안할 때 예산상의 절감효과도 상당히 거둘 수 있는 장점을 지니고 있다. 이렇듯 멀티플렉스 PCR 시스템이 가지는 인력, 예산 면에서의 효용가치로 인하여 유전자감식을 선도하는 영국, 미국을 비롯한 선진국가에서는 자국의 현실에 맞는 멀티플렉스 PCR 시스템을 확립하려는 연구가 활발히 이루어져 왔다.On the other hand, a multiplex PCR system is one in which a primer of several loci is inserted into a single PCR reaction and an experimental reaction is performed in a single tube. When a multiplex PCR system is used, taq Polymerase (PCR reaction enzyme) and other reagents, as well as minimizing the operation of the genotype capillary automatic analyzer, thereby reducing the cost. Gene detection has the advantage of being able to save considerably on budget considering that expensive commercial reagents are consumed. In this way, researches have been actively conducted to establish a multiplex PCR system suitable for the local reality in advanced countries such as the United Kingdom and the United States leading to gene identification due to the utility value of the multiplex PCR system.
프로메가(Promega Corporation, Madison, Wi, 미국)에서 상용 시판되고 있던 트리플렉스 PCR 키트(I, Ⅱ)에 근거하여 초기에는 트리플렉스 PCR 시스템 위주의 연구 개발이 활발히 이루어져 왔다(Kimpton et al.,1993; Urquhart et al., 1994). 그러나 4가지 유전좌위를 동시에 분석하는 콰드루플렉스 시스템(Sprecher et al., 1996 ; Lins et al., 1996 ; Robertson J.M et al., 1995)도 개발하기에 이르렀으며 현재는 그 이상의 유전좌위를 동시에 수행하는 멀티플렉스도 많은 연구가 이루어져 왔다(Kimpton et al., 1996; Evett et al.,1997).Based on the commercially available triplex PCR kit (I, II) commercially available from Promega Corporation (Madison, Wi, USA), research and development centered on triplex PCR systems have been actively conducted in the early days (Kimpton et al., 1993 Urquhart et al., 1994). However, the development of the Quadruplex system (Sprecher et al., 1996; Lins et al., 1996; Robertson JM et al., 1995), which simultaneously analyzes four genetic loci, (Kimpton et al., 1996; Yest et al., 1997) have also been performed.
최근 Thermofisher 사는 D3S1358, VWA, D16S539, CSF1PO, TPOX, Yindel, Amelogenin, D8S1179, D21S11, D18S51, DYS391, D2S441, D19S433, TH01, FGA, D22S1045, D5S818, D13S317, D7S820, SE33, D10S1248, D1S1656, D12S391, 및 D2S1338를 포함하는 24개 유전좌위의 동시 증폭 및 4색 검출 방법을 개발하였다(Globalfiler).In recent years, Thermofisher has developed and marketed D3S1358, VWA, D16S539, CSF1PO, TPOX, Yindel, Amelogenin, D8S1179, D21S11, D18S51, DYS391, D2S441, D19S433, TH01, FGA, D22S1045, D5S818, D13S317, D7S820, SE33, D10S1248, D1S1656, Simultaneous amplification and four-color detection of 24 genetic loci including D2S1338 have been developed (Globalfiler).
또한, 프로메가사는 D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, Amelogenin, VWA, D8S1179, TPOX, 및 FGA를 포함하는 16개 유전좌위의 동시 증폭 및 4색 검출 방법을 개발하였다(Powerplex 16).In addition, Promega was able to perform simultaneous amplification of 16 genetic loci including D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, Amelogenin, VWA, D8S1179, TPOX, 4-color detection method was developed (Powerplex 16).
이러한 멀티플렉스 PCR 시스템의 지속적인 개발성과는 DNA 데이터베이스(DNA intelligence database)의 설립 및 운용에 박차를 가할만한 획기적인 성과였다. 영국의 경우 1995년부터 대상자들의 입력을 시작한 이래로 2013년 3월 현재 6,700,000명 이상의 데이터를 보유하고 있으며 이 데이터베이스를 통하여 범인을 찾아낸 케이스는 평균 1주당 300건 이상의 범인을 색출해 내는 성과를 거두고 있다. 미국의 경우 과거 각 주 단위로 실시해 오던 데이터베이스는 미연방수사국(FBI)이 관리하는 CODIS(Combined DNA Index 시스템)을 이용하여 연방 전체의 국립 DNA 데이터베이스를 구축 통합하여 2013년 10월 현재 12,300,000명 이상의 대상자를 입력하여 매우 효율적으로 운용되고 있는 것으로 알려졌다. 그 외의 네덜란드 1996년, 오스트리아, 독일 1997년 또는 1998년 초에 대상자의 입력을 시작하여 현재 입력이 활발하게 이루어지고 상당한 효과를 거두고 있는 것으로 알려지고 있다. 한국에서도 2010년 8월 “DNA 신원확인 정보의 이용 및 보호에 관한 법률”이 발효되어 2014년 현재 173,024명의 대상자를 입력하였으며, 2014년 현재 4,252건의 사건을 해결하는데 결정적인 증거로 사용되었다고 알려져 있다.The continued development of such a multiplex PCR system has been a milestone in spurring the establishment and operation of a DNA database. As of March 2013, the UK has more than 6,700,000 data since the entry of the target population in 1995, and the database has been able to find out more than 300 criminals per week on average. In the United States, the database that has been conducted in each state in the past has been used to build and integrate a national DNA database of all federal states using CODIS (Combined DNA Index System) administered by the Federal Bureau of Investigation (FBI) And it is said to be operated very efficiently. Other Netherlands In 1996, Austria, Germany In 1997 or early 1998, it started to enter the subjects and it is known that the current input is active and has a considerable effect. In Korea, the "Act on the Use and Protection of DNA Identification Information" was enacted in August 2010, and 173,024 subjects were entered as of 2014, and it is known that it has been used as evidence for resolving 4,252 cases as of 2014.
현재 한국에서 널리 사용되고 있는 체세포 염색체 STR 증폭 방법 역시 외국회사에서 제조, 판매하고 있는 상용 키트이다. 이는 1990년대 후반 PCR에 의한 STR 분석법 개발을 주도하였던 미국, 영국에서 개발된 STR 마커들을 해당 국가의 회사들이 인수해 키트로 상용화하였기 때문이다. 특히 범죄자 DNA 데이터베이스가 세계적으로 일반화되면서 국가 간의 DNA 정보 교환을 위해 모든 국가가 공통적인 STR 마커를 포함하여 사용할 것이 권유되었고 이에 따라 후발국들은 모두 해당 STR 마커들을 광범위하게 포함하고 있는 상용화 키트를 사용하게 되었다.The somatic chromosome STR amplification method widely used in Korea is also a commercial kit manufactured and sold by a foreign company. This is because the STR markers developed in the US and UK, which led to the development of STR analysis method by PCR in the late 1990s, were acquired by the companies in the respective countries and commercialized as kits. In particular, as the criminal DNA database became globally universal, it was recommended that all countries use common STR markers for the exchange of DNA information between countries, so that later generations will use commercial kits that include a broad range of STR markers .
또한 위와 같은 상용화 키트는 편리하다는 장점도 있지만 외국회사의 기술에 의존하게 되는 단점을 지니고 있다. 실제로 이들 키트를 생산하는 회사는 앞서 살펴본 바와 같이 어플라이드 바이오시스템즈 社와 프로메가社가 주요 회사이며 이들 회사에서 10여 종의 상용 키트들이 생산 판매되고 있다. 세계시장을 독점하다시피한 이들 두 회사의 키트 가격은 매우 비싼 편으로 유전자 감식 비용의 큰 부분을 차지하고 있는 실정이다.In addition, the above-mentioned commercialization kit has a merit that it is convenient, but it has a disadvantage that it relies on foreign company's technology. In fact, the companies that produce these kits are, as we have seen, Applied Biosystems and Promega, which are a major company, with over 10 commercial kits being produced and sold. The kits for these two companies, which have dominated the global market, are very expensive and represent a large part of the cost of genetic testing.
KR 10-1672240호에서는 인간 객체 염색체 상의 STR 분석방법 및 이를 이용한 분석 키트를 개시하고 있고, KR 10-1667526호에서는 차세대 염기서열분석법을 이용한 인간 객체의 확장 상염색체 STR 분석방법을 개시하고 있으며, KR 10-0277289호에서는 한국인 집단에서 변별력을 가진 에스티알 유전자좌들로 구성된 콰드루플렉스 피씨알시스템 및 이를 이용한 유전자 감식방법을 개시하고 있고, KR 10-1008828호에서는 한국인 집단에서 변별력을 가진 16개의 짧은 반복 단위 유전좌위들 및 성염색체 유전좌위를 포함하는 멀티플렉스 유전자 증폭 시스템 및 이를 이용한 유전자 감식 방법을 개시하고 있으며, KR 10-1414827호에서는 멀티 플렉스 Y 성염색체 유전자 증폭 시스템을 이용한 유전자 감식 방법을 개시하고 있고, KR 10-1533792호에서는 NGS 기반 인간 객체의 상염색체 분석방법을 개시하고 있으며, KR 10-1457983호에서는 멀티플렉스 유전자 증폭을 이용한 분석대상의 인간 객체의 상염색체 분석 방법을 개시하고 있고, KR 10-0443569호에서는 길이다형성 DNA 염기서열을 이용한 다중 증폭중합효소연쇄반응 조건의 최적화에 의한 개인식별 및 친자감별 방법을 개시하고 있으며, KR 10-1341943호에서는 STR 검출용 키트 및 이를 이용한 STR 검출 방법을 개시하고 있고, KR 10-1716108호에서는 STR 유전좌위의 분별 선행 증폭을 통한 유전자 감식 방법을 개시하고 있으며, KR 10-1198096호에서는 개인식별 또는 친자확인을 하기 위한 특이 프라이머 및 이의 용도를 개시하고 있고, KR 10-2010-0081968호에서는 인간 X 염색체 짧은 연쇄반복 염기서열의 동시증폭 키트 및 방법을 개시하고 있으며, KR 10-2009-0111407호에서는 인간 성염색체 짧은 연쇄반복 염기서열의 동시증폭 키트 및 방법을 개시하고 있으며, US 9,090,943호에서는 PCR을 이용한 SE33(ACTBP2) 유전좌위 분석 방법을 개시하고 있고, US 9,797,841호에서는 SE33, PentaD, PentaE, D1S1656, D22S1045 및 D2S441이 포함된 STR 유전좌위의 대립유전자형을 결정하여 인각객체를 감식하는 방법을 개시하고 있다. 이러한 선행특허들의 마커 조합은 그 민감도와 정확도가 낮거나, NGS 등과 같은 복잡한 실험방법이 필요한 문제점이 있다.KR 10-1672240 discloses an STR analysis method on a human object chromosome and an assay kit using the same, and KR 10-1667526 discloses a method of analyzing an expanded human autologous chromosome STR using a next-generation sequencing method. KR 10-0277289 discloses a Quadruplex PCA system composed of Esterella loci with differentiating power in a Korean population and a gene detection method using the same, and KR 10-1008828 discloses 16 short repeats Unit genetic loci and sex chromosomal genetic loci, and a method for gene detection using the same, discloses a method of gene detection using a multiplex Y gene chromosome gene amplification system in KR 10-1414827 , And KR 10-1533792 discloses an autosomal analysis technique for NGS-based human subjects KR 10-1457983 discloses a method for analyzing human autosomes using a multiplex gene amplification method. In KR 10-0443569, a multiplex amplification polymerase Discloses a method of identifying a strand by discrimination of a strand and a strand discrimination method by optimizing a chain reaction condition. KR 10-1341943 discloses an STR detection kit and a STR detection method using the same, and in KR 10-1716108, Discloses a gene detection method using preamplification, KR 10-1198096 discloses a specific primer for identification or paternity identification and its use, and KR 10-2010-0081968 discloses a method of identifying a human X chromosome short chain repeat Discloses a kit and method for simultaneous amplification of a nucleotide sequence, and KR 10-2009-0111407 discloses a kit for the simultaneous amplification of a human chromosome short chain repeating base sequence US 9,090,943 discloses an analysis method of genetic loci of SE33 (ACTBP2) using PCR, and US 9,797,841 discloses a STR genetic library containing SE33, PentaD, PentaE, D1S1656, D22S1045 and D2S441. And determining the allelic genotype of the locus to identify the rat puppy object. The combination of the markers of these prior patents is problematic in that the sensitivity and accuracy are low, or that complex experimental methods such as NGS are required.
이에 본 발명자들은 민감도와 정확성이 높은 인간 객체의 유전자 감식을 위한 분석방법을 개발하기 위하여 예의 노력한 결과, 16개좌의 STR 유전좌 및 9개좌의 STR 유전좌를 듀얼 멀티플렉스 시스템으로 증폭할 경우, 추가적인 형광염료의 사용 없이 기존의 기계로 분석이 가능하면서 샘플의 유전정보를 높은 민감도와 정확도로 검출할 수 있음을 확인하고, 본 발명을 완성하였다. Accordingly, the present inventors have made intensive efforts to develop a method for analyzing a human subject having high sensitivity and accuracy. As a result, when amplifying a STR genome locus of 16 residues and a STR genome locus of 9 loci by a dual multiplex system, It is possible to perform the analysis with the conventional machine without using the fluorescent dye and to detect the genetic information of the sample with high sensitivity and accuracy, and the present invention has been completed.
본 배경기술 부분에 기재된 상기 정보는 오직 본 발명의 배경에 대한 이해를 향상시키기 위한 것이며, 이에 본 발명이 속하는 기술분야에서 통상의 지식을 가지는 자에게 있어 이미 알려진 선행기술을 형성하는 정보를 포함하지 않을 수 있다.The information described in the Background section is intended only to improve the understanding of the background of the present invention and thus does not include information forming a prior art already known to those skilled in the art .
발명의 요약SUMMARY OF THE INVENTION
본 발명의 목적은 멀티플렉스 유전자 증폭을 이용한 인간 객체의 유전자 감식을 위한 분석 방법을 제공하는 것이다.It is an object of the present invention to provide an analysis method for gene detection of a human object using multiplex gene amplification.
본 발명의 다른 목적은 24개 유전좌를 증폭할 수 있는 프라이머 세트를 포함하는 인간 객체(human subject) 염색체 상의 STR (short tandem repeat) 분석용 멀티플렉스 유전자 증폭 키트를 제공하는 것이다.Another object of the present invention is to provide a multiplex gene amplification kit for STR (short tandem repeat) analysis on a human subject chromosome comprising a set of primers capable of amplifying 24 genetic loci.
상기 목적을 달성하기 위해서, 본 발명은In order to achieve the above object,
(a) D8S1179, CSF1PO(Human c-fms protooncogene for CSF-1 receptor gene), D3S1358, D2S1338, 아멜로제닌(Amelogenin), vWA(von Willebrand factor A), TH01, D18S51, D5S818, D21S11, TPOX(Human thyroid peroxidase gene), FGA(Human fibrinogen alpha chain), D13S317, D7S820, D16S539 및 D19S433를 포함하는 유전좌위 각각을 증폭할 수 있는 프라이머를 포함하는 제1프라이머 세트; 및 아멜로제닌, SE33, D1S1656, Penta D, D22S1045, D10S1248, D12S391, D2S441 및 Penta E를 포함하는 유전자 각각을 증폭할 수 있는 프라이머를 포함하는 제2프라이머 세트와 인간 객체 DNA 시료를 각각 독립적으로 반응시켜 증폭시키는 단계; 및(a) D8S1179, CSF1PO (Human c-fms protooncogene for CSF-1 receptor gene), D3S1358, D2S1338, Amelogenin, vWA (von Willebrand factor A), TH01, D18S51, D5S818, D21S11, TPOX thyroid peroxidase gene), a human fibrinogen alpha chain (FGA), D13S317, D7S820, D16S539 and D19S433; And a second primer set comprising primers capable of amplifying each of the genes including amelogenin, SE33, D1S1656, Penta D, D22S1045, D10S1248, D12S391, D2S441 and Penta E and a human primate set ; And
(b) 상기 단계 (a)의 멀티플렉스 증폭 산물을 이용하여 상기 유전좌위의 대립유전자형을 결정하는 단계;를 포함하는 멀티플렉스 유전자 증폭을 이용한 인간 객체의 유전자 감식을 위한 분석방법을 제공한다. and (b) determining an allele of the genetic locus using the multiplex amplification product of step (a). The present invention also provides an assay method for gene detection of a human object using multiplex gene amplification.
본 발명은 또한, (a) D8S1179, CSF1PO(Human c-fms protooncogene for CSF-1 receptor gene), D3S1358, D2S1338, 아멜로제닌(Amelogenin), vWA(von Willebrand factor A), TH01, D18S51, D5S818, D21S11, TPOX(Human thyroid peroxidase gene), FGA(Human fibrinogen alpha chain), D13S317, D7S820, D16S539 및 D19S433를 포함하는 유전좌위 각각을 증폭할 수 있는 프라이머를 포함하는 제1프라이머 세트; 및 아멜로제닌, SE33, D1S1656, Penta D, D22S1045, D10S1248, D12S391, D2S441 및 Penta E를 포함하는 유전자 각각을 증폭할 수 있는 프라이머를 포함하는 제2프라이머 세트와 인간 객체 DNA 시료를 각각 독립적으로 반응시켜 증폭시키는 단계; 및The present invention also provides a pharmaceutical composition comprising (a) D8S1179, CSF1PO (Human c-fms protooncogene for CSF-1 receptor gene), D3S1358, D2S1338, Amelogenin, von Willebrand factor A, TH01, D18S51, D5S818, A first primer set comprising primers capable of amplifying each of the genetic loci comprising D21S11, human thyroid peroxidase gene (TPOX), human fibrinogen alpha chain (FGA), D13S317, D7S820, D16S539 and D19S433; And a second primer set comprising primers capable of amplifying each of the genes including amelogenin, SE33, D1S1656, Penta D, D22S1045, D10S1248, D12S391, D2S441 and Penta E and a human primate set ; And
(b) 상기 단계 (a)의 멀티플렉스 증폭 산물을 이용하여 상기 유전좌위의 대립유전자형을 결정하는 단계;를 포함하는 멀티플렉스 유전자 증폭을 이용한 인간 객체의 유전자 감식방법을 제공한다.(b) determining an allele of the genetic locus using the multiplex amplification product of step (a).
본 발명은 또한, D8S1179, CSF1PO(Human c-fms protooncogene for CSF-1 receptor gene), D3S1358, D2S1338, 아멜로제닌(Amelogenin), vWA(von Willebrand factor A), TH01, D18S51, D5S818, D21S11, TPOX(Human thyroid peroxidase gene), FGA(Human fibrinogen alpha chain), D13S317, D7S820, D16S539 및 D19S433를 포함하는 유전좌위 각각을 증폭할 수 있는 프라이머를 포함하는 제1프라이머 세트; 및 아멜로제닌, SE33, D1S1656, Penta D, D22S1045, D10S1248, D12S391, D2S441 및 Penta E를 포함하는 유전자 각각을 증폭할 수 있는 프라이머를 포함하는 제2프라이머 세트를 포함하는 인간 객체(human subject) 염색체 상의 STR (short tandem repeat) 분석용 멀티플렉스 유전자 증폭 키트를 제공한다.The present invention also relates to the use of a polynucleotide selected from the group consisting of D8S1179, CSF1PO (Human c-fms protooncogene for CSF-1 receptor gene), D3S1358, D2S1338, Amelogenin, von Willebrand factor A, TH01, D18S51, D5S818, D21S11, (Human thyroid peroxidase gene), a human fibrinogen alpha chain (FGA), D13S317, D7S820, D16S539 and D19S433; And a second primer set comprising a primer capable of amplifying each of the genes including amelogenin, SE33, D1S1656, Penta D, D22S1045, D10S1248, D12S391, D2S441 and Penta E, A multiplex gene amplification kit for analyzing short tandem repeat (STR)
도 1은 본 발명에 따른 듀얼 멀티플렉스 시스템에서 분석하는 유전좌의 위치를 형광 마커별로 표시한 개략도이다.FIG. 1 is a schematic view showing positions of a genetic locus analyzed in a dual multiplex system according to the present invention for each fluorescent marker.
도 2는 본 발명에 따른 듀얼 멀티플렉스 시스템에서 제1 프라이머 세트를 이용한 샘플 분석 결과이다.2 shows a result of a sample analysis using a first primer set in a dual multiplex system according to the present invention.
도 3은 본 발명에 따른 듀얼 멀티플렉스 시스템에서 제2 프라이머 세트를 이용한 샘플 분석 결과이다.3 shows a result of a sample analysis using a second primer set in a dual multiplex system according to the present invention.
발명의 상세한 설명 및 바람직한 구현예DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로 본 명세서에서 사용된 명명법은 본 기술분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.
본 발명에서의 용어 'STR'은 인간 게놈(genome) 내의 어떤 형질 등의 유전정보를 담고 있지 않은 인트론(intron) 내에 널리 분포한 것으로 알려진 반복염기서열(tandem repeat sequence)들은 유전자 감정의 마커(marker)로서 범세계적으로 유용하게 사용되고 있다. 인간의 게놈에서 많은 유전좌위가 다형성의 STR 부위를 함유하고 있다. STR 좌위는 길이가 2 내지 7개의 염기쌍인 짧은 반복 서열 요소로 구성되어 있는데, 인간의 게놈에는 매 15 kb마다 한 번씩, 2백 만개의 삼량체 및 사량체 STR이 존재하는 것으로 추정된다. 특정 좌위에서의 짧은 반복 배열 단위의 수가 변함에 따라, 이 좌위에서의 DNA 길이가 각 대립 형질(allele) 및 각 개인에따라 변하게 된다. STR 좌위는 이 반복 배열의 측부에 동정되어 있는 특정 프라이머 서열을 이용하여 폴리머라제 연쇄 반응법(PCR)을 통해 증폭시킬 수 있다.The term 'STR' in the present invention refers to a tandem repeat sequence known to be widely distributed within an intron that does not contain any genetic information such as a trait in the human genome, ), Which has been widely used worldwide. Many genetic loci in the human genome contain the STR region of the polymorphism. The STR locus consists of a short repeat sequence element, 2 to 7 base pairs in length, which is estimated to have 2 million trimer and tetramer STRs once every 15 kb in the human genome. As the number of short repeating units in a particular locus changes, the DNA length at that locus will vary with each allele and each individual. STR loci can be amplified by Polymerase Chain Reaction (PCR) using specific primer sequences identified on the side of this repeat sequence.
이러한 유전좌위의 대립 형질들은 증폭된 부위 내에 함유되어 있는 반복 서열의 복제수(the number of copy)에 따라 구분되며, 전기 영동법으로 분리한 후, 방사성, 형광, 실버 스테인 및 색채 등 적당한 검출 방법을 사용하여 식별한다.These alleles of the genetic loci are classified according to the number of copies of the repeated sequences contained in the amplified region. After separation by electrophoresis, suitable detection methods such as radioactive, fluorescent, silver stain and color .
본 발명에서 용어 ‘증폭’은 핵산 분자를 증폭하는 반응을 의미한다. 다양한 증폭 반응들이 당업계에 보고 되어 있으며, 이는 중합효소 연쇄반응(이하 PCR이라 한다)(미국 특허 제4,683,195, 4,683,202, 및 4,800,159호), 역전사-중합효소 연쇄반응(이하 RT-PCR로 표기한다), WO 89/06700 및 EP 329,822의 방법, 리가아제 연쇄 반응(ligase chain reaction; LCR, WO 90/01069), 복구 연쇄 반응(repair chain reaction; EP 439,182), 전사-중재 증폭(transcription-mediated amplification; MA, WO 88/10315), 자가 유지 염기서열 복제(self sustained sequence replication, WO 90/06995), 타깃 폴리뉴클레오티드 염기서열의 선택적 증폭(selective amplification of target polynucleotide sequences, 미국 특허 제6,410,276호), 컨센서스 서열 프라이밍 중합효소 연쇄 반응(consensus sequence primed polymerase chain reaction; CP-PCR, 미국 특허 제4,437,975호), 임의적 프라이밍 중합효소 연쇄 반응(arbitrarily primed polymerase chain reaction; AP-PCR, 미국 특허 제5,413,909호 및 제5,861,245호), 핵산 염기서열 기반 증폭(nucleic acid sequence based amplification; NASBA, 미국 특허 제5,130,238호, 제5,409,818호, 제5,554,517호, 및 제6,063,603호), 가닥 치환 증폭(strand displacement amplification) 및 고리-중재 항온성 증폭(loop-mediated isothermal amplification; LAMP)을 포함하나, 이에 한정되지는 않는다.The term " amplification " in the present invention means a reaction for amplifying a nucleic acid molecule. A variety of amplification reactions have been reported in the art, including polymerase chain reaction (PCR) (US Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), reverse transcription-polymerase chain reaction (RT- , The methods of WO 89/06700 and EP 329,822, ligase chain reaction (LCR, WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification MA, WO 88/10315), self sustained sequence replication (WO 90/06995), selective amplification of target polynucleotide sequences (US Pat. No. 6,410,276), consensus sequence PCR was performed using the primer polymerase chain reaction (CP-PCR, US Pat. No. 4,437,975), arbitrary primed polymerase chain reaction (AP-PCR, 5,413,909 and 5,861,245), nucleic acid sequence based amplification (NASBA, U.S. Pat. Nos. 5,130,238, 5,409,818, 5,554,517 and 6,063,603), strand displacement but are not limited to, amplification and loop-mediated isothermal amplification (LAMP).
사용 가능한 다른 증폭 방법들은 미국특허 제5,242,794, 5,494,810, 4,988,617호 및 미국 특허 제09/854,317호에 기술되어 있다.Other amplification methods that may be used are described in U.S. Patent Nos. 5,242,794, 5,494,810, 4,988,617 and U.S. Patent No. 09 / 854,317.
PCR은 가장 잘 알려진 핵산 증폭 방법으로, 그의 많은 변형과 응용들이 개발되어 있다. 예를 들어, PCR의 특이성 또는 민감성을 증진시키기 위해 전통적인 PCR 절차를 변형시켜 터치다운(touchdown) PCR, 핫 스타트(hot start) PCR, 네스티드(nested) PCR 및 부스터(booster) PCR이 개발되었다. 또한, 실시간(real-time) PCR, 분별 디스플레이 PCR(differential display PCR, D-PCR), cDNA 말단의 신속 증폭(rapid amplification of cDNA ends, RACE), DL-PCR(PC), 인버스 중합효소 연쇄반응(inverse polymerase chain reaction: IPCR), 벡토레트(vectorette) PCR, 및 TAIL-PCR(thermal asymmetric interlaced PCR)이 특정한 응용을 위해 개발되었다. PCR에 대한 자세한 내용은 McPherson, M.J., 및 Moller, S.G. PCR. BIOS Scientific Publishers, Springer-Verlag New York Berlin Heidelberg, N.Y. (2000)에 기재되어 있으며, 그의 교시사항은 본 명세서에 참조로 삽입된다.PCR is the most well-known nucleic acid amplification method, and many variations and applications thereof have been developed. For example, touchdown PCR, hot start PCR, nested PCR and booster PCR have been developed by modifying traditional PCR procedures to enhance the specificity or sensitivity of PCR. In addition, real-time PCR, differential display PCR, D-PCR, rapid amplification of cDNA ends (RACE), DL-PCR (PC), inverse polymerase chain reaction inverse polymerase chain reaction (IPCR), vectorette PCR, and thermal asymmetric interlaced PCR (TAIL-PCR) have been developed for specific applications. For more information on PCR see McPherson, M.J., and Moller, S.G. PCR. BIOS Scientific Publishers, Springer-Verlag New York Berlin Heidelberg, N.Y. (2000), the teachings of which are incorporated herein by reference.
상기 멀티플렉스 증폭은 멀티플렉스 PCR(Polymerase Chain Reaction) 증폭이다. 본 발명의 일 구현예에 따르면, 상기 멀티플렉스 PCR 증폭은 57-61℃의 어닐링(annealing) 온도 조건을 갖고, 본 발명의 다른 구현예에 따르면, 상기 멀티플렉스 PCR 증폭은 58-60℃의 어닐링 온도 조건을 가지며, 본 발명의 특정 구현예에 따르면, 상기 멀티플렉스 PCR 증폭은 58.5-59.5℃의 어닐링 온도 조건을 갖는다.The multiplex amplification is a multiplex PCR (Polymerase Chain Reaction) amplification. According to one embodiment of the present invention, the multiplex PCR amplification has an annealing temperature condition of 57-61 ° C. According to another embodiment of the present invention, the multiplex PCR amplification is performed at a temperature of 58-60 ° C Temperature conditions, and according to certain embodiments of the present invention, the multiplex PCR amplification has an annealing temperature condition of 58.5-59.5 [deg.] C.
상기 멀티플렉스 PCR 증폭은 PCR을 실시하는 데 적정한 싸이클 수가 요구된다. 본 발명의 일 구현예에 따르면, 상기 멀티플렉스 PCR 증폭은 27-29 싸이클로 실시한다. 본 발명의 멀티플렉스 PCR 증폭을 26 싸이클 이하로 실시하는 경우에 500 RFU 이하의 피크들이 형성되었고, 30 싸이클에서는 2,000 RFU 이상의 피크가 형성되었지만 노이즈가 증가하고 불완전한 A 삽입이 발생하여 적합하지 않다.The multiplex PCR amplification requires a reasonable number of cycles to perform PCR. According to one embodiment of the invention, the multiplex PCR amplification is performed in 27-29 cycles. When the multiplex PCR amplification of the present invention was carried out in 26 cycles or less, peaks of 500 RFU or less were formed and peaks of 2,000 RFU or more were formed in 30 cycles, but noise was increased and incomplete A insertion occurred.
본 발명에서 용어 ‘듀얼 멀티플렉스 시스템’은 하나의 샘플에서 수득한 핵산 시료를 각각의 독립된 프라이머 세트를 이용하여, 멀티플렉스 시스템을 동시에 2회 이상 수행하는 것을 의미한다.The term " dual multiplex system " in the present invention means that a nucleic acid sample obtained from one sample is simultaneously subjected to multiplex system two or more times using each independent primer set.
본 발명에서 용어 ‘프라이머(primer)’는 적합한 온도에서 적합한 완충액 내에서 적합한 조건(즉, 4종의 다른 뉴클레오시드트리포스페이트 및 중합 반응 효소 하에서 주형-지시 DNA 합성의 개시점으로 작용할 수 있는 단일 가닥의 올리고뉴클레오티드를 의미한다. 프라이머의 적합한 길이는 다양한 인자, 예를 들어, 온도와 프라이머의 용도에 따라 차이가 있지만 전형적으로 15 내지 30개의 뉴클레오티드이다. 짧은 프라이머는 주형과 충분히 안정된 혼성화 복합체를 형성하기 위하여 일반적으로 보다 낮은 온도를 요구할 수 있다. 용어 "전방향 프라이머(forward primer)" 및 "역방향 프라이머(reverse primer)"는 중합 효소 연쇄 반응에 의해 증폭되는 주형의 일정한 부위의 3' 말단 및 5' 말단에 각각 결합하는 프라이머를 의미한다. 프라이머의 서열은 주형의 일부 서열과 완전하게 상보적인 서열을 가질 필요는 없으며, 주형과 혼성화 되어 프라이머 고유의 작용을 할 수 있는 범위 내에서의 충분한 상보성을 가지면 충분하다. 따라서, 일 구체예에 따른 프라이머 세트는 주형인 뉴클레오티드 서열에 완벽하게 상보적인 서열을 가질 필요는 없으며, 이 서열에 혼성화되어 프라이머 작용을 할 수 있는 범위 내에서 충분한 상보성을 가지면 충분한 것으로 해석된다. 이러한 프라이머의 디자인은 주형이 되는 폴리뉴클레오티드의 염기 서열을 참조하여 당업자에 의해 용이하게 실시할 수 있으며, 예를 들어, 프라이머 디자인용 프로그램(예를 들어, PRIMER 3, VectorNTI 프로그램)을 이용하여 할 수 있다.The term " primer " as used herein refers to a primer that is capable of hybridizing under appropriate conditions (i.e., a single nucleotide which can act as a starting point for template-directed DNA synthesis under four different nucleoside triphosphates and polymerase, The appropriate length of the primer is typically 15 to 30 nucleotides, depending on various factors, for example, temperature and use of the primer. The short primer forms a sufficiently stable hybridization complex with the template. The term "forward primer" and "reverse primer" refer to the 3 'end of a constant region of a template amplified by a polymerase chain reaction and the 5' The term " primer " refers to a part of the template. It is not necessary to have a sequence completely complementary to the heat, and it is sufficient that the primer set has sufficient complementarity within a range capable of hybridizing with a template and acting as a primer. Therefore, a set of primers according to one embodiment includes a nucleotide sequence It is not necessary to have a perfectly complementary sequence, and it is considered sufficient if it has sufficient complementarity within a range that it can hybridize to this sequence to give primer action. The design of such a primer refers to the nucleotide sequence of a polynucleotide to be a template Can be easily carried out by a person skilled in the art, for example, by using a program for primer design (for example, PRIMER 3, VectorNTI program).
본 발명에서는 STR 유전좌의 위치를 듀얼 멀티플렉스 시스템을 이용하여 분석할 경우, 형광염료를 추가로 사용하지 않으면서 분석 민감도와 정확도가 증가하는 지를 확인하고자 하였다.In the present invention, when analyzing the position of the STR genetic locus using a dual multiplex system, it was tried to confirm whether the sensitivity and accuracy of the assay increase without using a fluorescent dye.
즉, 본 발명의 일 실시예에서는 인간 객체 DNA 시료를 D8S1179, CSF1PO(Human c-fms protooncogene for CSF-1 receptor gene), D3S1358, D2S1338, 아멜로제닌(Amelogenin), vWA(von Willebrand factor A), TH01, D18S51, D5S818, D21S11, TPOX(Human thyroid peroxidase gene), FGA(Human fibrinogen alpha chain), D13S317, D7S820, D16S539 및 D19S433으로 구성된 유전좌위 각각을 증폭할 수 있는 프라이머를 포함하는 제1프라이머 세트; 및 아멜로제닌, SE33, D1S1656, Penta D, D22S1045, D10S1248, D12S391, D2S441 및 Penta E로 구성된 유전자 각각을 증폭할 수 있는 프라이머를 포함하는 제2프라이머 세트와 각각 독립적으로 반응시켜 증폭을 실시한 다음, 각 유전좌의 대립유전자형을 결정하여 감식한 결과, 높은 민감도와 정확도로 인간 객체의 유전자를 감식할 수 있음을 확인하였다(도 2, 3).That is, in one embodiment of the present invention, a human object DNA sample is treated with D8S1179, CSF1PO (Human c-fms protooncogene for CSF-1 receptor gene), D3S1358, D2S1338, Amelogenin, vWA (von Willebrand factor A) A first primer set comprising primers capable of amplifying respective genetic loci composed of TH01, D18S51, D5S818, D21S11, human thyroid peroxidase gene (TPOX), human fibrinogen alpha chain (FGA), D13S317, D7S820, D16S539 and D19S433; And a second primer set containing primers capable of amplifying each of the genes composed of amelogenin, SE33, D1S1656, Penta D, D22S1045, D10S1248, D12S391, D2S441 and Penta E, As a result of determining allele genotypes of each genetic locus, it was confirmed that genes of human object can be identified with high sensitivity and accuracy (FIGS. 2 and 3).
따라서, 본 발명은 일 관점에서, (a) D8S1179, CSF1PO(Human c-fms protooncogene for CSF-1 receptor gene), D3S1358, D2S1338, 아멜로제닌(Amelogenin), vWA(von Willebrand factor A), TH01, D18S51, D5S818, D21S11, TPOX(Human thyroid peroxidase gene), FGA(Human fibrinogen alpha chain), D13S317, D7S820, D16S539 및 D19S433를 포함하는 유전좌위 각각을 증폭할 수 있는 프라이머를 포함하는 제1프라이머 세트; 및 아멜로제닌, SE33, D1S1656, Penta D, D22S1045, D10S1248, D12S391, D2S441 및 Penta E를 포함하는 유전자 각각을 증폭할 수 있는 프라이머를 포함하는 제2프라이머 세트와 인간 객체 DNA 시료를 각각 독립적으로 반응시켜 증폭시키는 단계; 및 (b) 상기 단계 (a)의 멀티플렉스 증폭 산물을 이용하여 상기 유전좌위의 대립유전자형을 결정하는 단계;를 포함하는 멀티플렉스 유전자 증폭을 이용한 인간 객체의 유전자 감식을 위한 분석방법에 관한 것이다.Accordingly, the present invention provides, in one aspect, (a) a compound selected from the group consisting of D8S1179, CSF1PO (human c-fms protooncogene for CSF-1 receptor gene), D3S1358, D2S1338, Amelogenin, vWA, A first primer set comprising primers capable of amplifying each of the genetic loci comprising D18S51, D5S818, D21S11, Human thyroid peroxidase gene (TPOX), Human fibrinogen alpha chain (FGA), D13S317, D7S820, D16S539 and D19S433; And a second primer set comprising primers capable of amplifying each of the genes including amelogenin, SE33, D1S1656, Penta D, D22S1045, D10S1248, D12S391, D2S441 and Penta E and a human primate set ; And (b) determining an allelic genotype of the genetic locus using the multiplex amplification product of step (a). The present invention relates to a method for gene detection of a human object using multiplex gene amplification.
본 발명은 다른 관점에서, 본 발명은 또한, (a) D8S1179, CSF1PO(Human c-fms protooncogene for CSF-1 receptor gene), D3S1358, D2S1338, 아멜로제닌(Amelogenin), vWA(von Willebrand factor A), TH01, D18S51, D5S818, D21S11, TPOX(Human thyroid peroxidase gene), FGA(Human fibrinogen alpha chain), D13S317, D7S820, D16S539 및 D19S433를 포함하는 유전좌위 각각을 증폭할 수 있는 프라이머를 포함하는 제1프라이머 세트; 및 아멜로제닌, SE33, D1S1656, Penta D, D22S1045, D10S1248, D12S391, D2S441 및 Penta E를 포함하는 유전자 각각을 증폭할 수 있는 프라이머를 포함하는 제2프라이머 세트와 인간 객체 DNA 시료를 각각 독립적으로 반응시켜 증폭시키는 단계; 및 (b) 상기 단계 (a)의 멀티플렉스 증폭 산물을 이용하여 상기 유전좌위의 대립유전자형을 결정하는 단계;를 포함하는 멀티플렉스 유전자 증폭을 이용한 인간 객체의 유전자 감식방법에 관한 것이다.In another aspect, the present invention is directed to a method for modulating the expression of a protein selected from the group consisting of (a) D8S1179, CSF1PO (Human c-fms protooncogene for CSF-1 receptor gene), D3S1358, D2S1338, Amelogenin, vWA , Primers capable of amplifying each of the genetic loci including TH01, D18S51, D5S818, D21S11, human thyroid peroxidase gene (TPOX), human fibrinogen alpha chain (FGA), D13S317, D7S820, D16S539 and D19S433 set; And a second primer set comprising primers capable of amplifying each of the genes including amelogenin, SE33, D1S1656, Penta D, D22S1045, D10S1248, D12S391, D2S441 and Penta E and a human primate set ; And (b) determining an allele of the genetic locus using the multiplex amplification product of step (a). 2. Description of the Related Art
본 발명에 있어서, 상기 프라이머는 각 STR좌를 증폭할 수 있고, GC 비율이 40 내지 60% 범위를 유지하며, 종열반복 배열이 없는 유전자 서열과 상보적인 것을 특징으로 할 수 있다.In the present invention, the primer may be characterized by being capable of amplifying STR strands, maintaining the GC ratio in the range of 40 to 60%, and complementary to the gene sequence having no repeating sequence of nucleotides.
본 발명에 있어서, 상기 제1프라이머 세트는 서열번호 1 내지 32의 염기서열로 표시되는 것을 특징으로 할 수 있다.In the present invention, the first primer set may be characterized by being represented by the nucleotide sequences of SEQ ID NOS: 1-32.
본 발명에 있어서, 상기 제2프라이머 세트는 서열번호 33 내지 50의 염기서열로 표시되는 프라이머인 것을 특징으로 할 수 있다.In the present invention, the second primer set may be a primer represented by the nucleotide sequence of SEQ ID NOS: 33 to 50.
본 발명에 있어서, 상기 (a) 단계의 증폭산물은 80 내지 400bp인 것을 특징으로 할 수 있다. In the present invention, the amplification product of step (a) may be 80 to 400 bp.
본 발명에 있어서, 상기 단계 (b)는 상기 멀티플렉스 증폭 산물에서 증폭된 대립유전자의 크기를 크기 표준물(size standard)과 비교/평가하여 실시하며, 상기 크기 표준물은 DNA 마커 또는 유전좌위-특이적 대립유전자 래더인 것을 특징으로 할 수 있다.In the present invention, step (b) is performed by comparing the size of the allele amplified in the multiplex amplification product with a size standard, and the size standard is a DNA marker or a genetic locus- Specific allele gene ladder.
본 발명에 있어서, 상기 DNA 시료는 혈액, 정액, 질 세포, 모발, 타액, 소변, 구강세포, 태반세포 또는 태아세포를 포함하는 양수 및 이의 혼합물을 포함하는 군으로부터 선택되는 조직으로부터 분리된 DNA 시료인 것을 특징으로 할 수 있다.In the present invention, the DNA sample may be a DNA sample isolated from a tissue selected from the group consisting of blood, semen, vaginal cells, hair, saliva, urine, oral cells, placental cells or fetal cells, .
본 발명에 있어서, 상기 생체 시료는 혈액, 모발, 타액, 소변, 구강세포 및 이들의 혼합물로 구성된 군으로부터 선택되는 것을 특징으로 할 수 있다.In the present invention, the biological sample may be selected from the group consisting of blood, hair, saliva, urine, oral cells, and mixtures thereof.
본 발명에 있어서, 인간 객체의 유전자 감식은 대립유전자형을 결정하고, 이를 대조군과 비교하여 객체의 유전자 형을 결정하는 것을 의미한다. 예를 들어, 본 발명의 방법으로 범죄 현장에서 발견된 혈액 샘플 내의 DNA의 유전자 감식을 수행할 경우, 결정된 대립유전자형이 범죄자 인터페이스에 존재하는 지 여부를 판단하여, 용의자를 특정할 수 있으며, 친자확인에 이용할 경우, 샘플 객체의 대립유전자형을 결정하고 이를 친부 또는 친보의 대립유전자형을 대조군으로 비교하여 친자 여부를 확인하는 것을 의미한다.In the present invention, gene detection of a human object means determining the genotype of an object by determining an allele genotype and comparing it with a control. For example, when gene detection of DNA in a blood sample found at a crime scene is performed by the method of the present invention, it is possible to determine whether the determined allelotype exists in the criminal interface, to identify the suspect, , It means to determine the allelic genotype of the sample object, and to compare the allelic genotype of the father or the progeny with the control group to confirm the parental status.
본 발명에 있어서, 상기 제1프라이머 세트 및 제2프라이머 세트는 형광염료로 표지되는 것을 특징으로 할 수 있다.In the present invention, the first primer set and the second primer set may be labeled with a fluorescent dye.
본 발명에서, 상기 서열번호 1 내지 8 및 서열번호 33 내지 34의 염기서열로 표시되는 프라이머 세트는 제1형광염료로 표지되고, 상기 서열번호 9 내지 16 및 서열번호 35 내지 40의 염기서열로 표시되는 프라이머 세트는 제2형광염료료 표지되며, 상기 서열번호 17 내지 24 및 서열번호 41 내지 46의 염기서열로 표시되는 프라이머 세트는 제3형광염료로 표지되고, 상기 서열번호 25 내지 32 및 서열번호 47 내지 50의 염기서열로 표시되는 프라이머 세트는 제4형광염료로 표지되는 것을 특징으로 할 수 있다.In the present invention, the primer set represented by the nucleotide sequences of SEQ ID NOS: 1 to 8 and SEQ ID NOS: 33 to 34 is labeled with the first fluorescent dye, and the primers set forth in the nucleotide sequences of SEQ ID NOS: 9 to 16 and SEQ ID NOS: And a primer set represented by the nucleotide sequences of SEQ ID NOS: 17 to 24 and SEQ ID NOS: 41 to 46 are labeled with a third fluorescent dye, and the primer sets set forth in SEQ ID NOS: 25 to 32 and SEQ ID NOS: The primer set represented by the nucleotide sequence of 47 to 50 can be characterized as being labeled with a fourth fluorescent dye.
본 발명의 유전자 감식 방법에 이용되는 프라이머는 유전좌위에 상보적으로 결합하는 한 쌍의 프라이머 중 하나의 프라이머의 5’말단에 형광염료가 표지된다. 상기 제1형광염료 내지 제4형광염료는 모세관 전기영동을 통해 검출할 수 있도록 한 레인에 3 내지 4개의 유전좌위를 배치하는 구성하기 위한 표지이다.In the primer used in the gene detection method of the present invention, a fluorescent dye is labeled at the 5 'end of one primer of a pair of primers complementarily binding to the genetic locus. The first to fourth fluorescent dyes are labels for constituting three to four genetic loci in a lane so that they can be detected through capillary electrophoresis.
본 발명에 있어서, 상기 형광염료는 6-FAM, VIC, NED, PET, ROX, HEX, dR110, dR6G, dTAMRA 및 dROX로 구성된 군으로부터 선택되는 하나 이상일 수 있으나, 이에 한정하지는 않는다.In the present invention, the fluorescent dye may be at least one selected from the group consisting of 6-FAM, VIC, NED, PET, ROX, HEX, dR110, dR6G, dTAMRA and dROX.
본 발명의 유전자 감식 방법에 이용되는 프라이머는 피크 균형을 맞추기 위한 최적의 배합비를 갖는다.The primer used in the gene detection method of the present invention has an optimal blending ratio for adjusting the peak balance.
본 발명의 일 실시예에 따르면, 제1프라이머 세트에서 D8S1179 유전좌위에 상보적으로 결합하는 프라이머는 1.0-5.0μM의 최종 농도를 가지고, CSF1PO 유전좌위에 상보적으로 결합하는 프라이머는 0.1-2.0μM의 최종 농도를 가지고, D3S1358 유전좌위에 상보적으로 결합하는 프라이머는 0.1-2.0μM의 최종 농도를 가지고, D2S1338 유전좌위에 상보적으로 결합하는 프라이머는 0.5-3.0μM의 최종 농도를 가지고, Amelogenin 유전좌위에 상보적으로 결합하는 프라이머는 0.1-2.0μM의 최종 농도를 가지고, vWA 유전좌위에 상보적으로 결합하는 프라이머는 1.0-5.0μM의 최종 농도를 가지고, TH01 유전좌위에 상보적으로 결합하는 프라이머는 0.1-2.0μM의 최종 농도를 가지고, D18S51 유전좌위에 상보적으로 결합하는 프라이머는 0.1-2.0μM의 최종 농도를 가지고, D5S818 유전좌위에 상보적으로 결합하는 프라이머는 1.0-5.0μM의 최종 농도를 가지고, D21S11 유전좌위에 상보적으로 결합하는 프라이머는 5.0-20.0μM의 최종 농도를 가지고, TPOX 유전좌위에 상보적으로 결합하는 프라이머는 0.1-2.0μM의 최종 농도를 가지고, FGA 유전좌위에 상보적으로 결합하는 프라이머는 0.5-3.0μM의 최종 농도를 가지고, D13S317 유전좌위에 상보적으로 결합하는 프라이머는 2.0-8.0μM의 최종 농도를 가지고, D7S820 유전좌위에 상보적으로 결합하는 프라이머는 0.5-3.0μM의 최종 농도를 가지고, D16S539 유전좌위에 상보적으로 결합하는 프라이머는 2.0-8.0μM의 최종 농도를 가지고, D19S433 유전좌위에 상보적으로 결합하는 프라이머는 0.5-3.0μM의 최종 농도를 가지며, 제2프라이머 세트에서 SE33 유전좌위에 상보적으로 결합하는 프라이머는 0.1-2.0μM의 최종 농도를 가지고, Amelogenin 유전좌위에 상보적으로 결합하는 프라이머는 0.1-2.0μM의 최종 농도를 가지고, D1S1656 유전좌위에 상보적으로 결합하는 프라이머는 0.1-2.0μM의 최종 농도를 가지고, Penta D 유전좌위에 상보적으로 결합하는 프라이머는 0.1-2.0μM의 최종 농도를 가지고, D22S1045 유전좌위에 상보적으로 결합하는 프라이머는 0.1-2.0μM의 최종 농도를 가지고, D10S1248 유전좌위에 상보적으로 결합하는 프라이머는 0.5-3.0μM의 최종 농도를 가지고, D12S391 유전좌위에 상보적으로 결합하는 프라이머는 0.5-3.0μM의 최종 농도를 가지고, D2S441 유전좌위에 상보적으로 결합하는 프라이머는 0.1-2.0μM의 최종 농도를 가지고, Penta E 유전좌위에 상보적으로 결합하는 프라이머는 0.5-3.0μM의 최종 농도를 가지는 것을 특징으로 할 수 있다.According to one embodiment of the invention, the primer that binds complementarily to the D8S1179 genetic locus in the first primer set has a final concentration of 1.0-5.0 [mu] M and the primer that binds complementarily to the CSF1PO genetic locus is 0.1-2.0 [mu] M Primers complementarily binding to the D3S1358 genetic locus have a final concentration of 0.1-2.0 [mu] M, primers that bind complementarily to the D2S1338 genetic locus have a final concentration of 0.5-3.0 [mu] M, the Amelogenin genetic Primers that complementarily bind to the locus have a final concentration of 0.1-2.0 [mu] M, primers that bind complementarily to the vWA genetic locus have a final concentration of 1.0-5.0 [mu] M, primers that complementarily bind to the TH01 genetic locus Has a final concentration of 0.1-2.0 [mu] M, the primer that binds complementarily to the D18S51 genetic locus has a final concentration of 0.1-2.0 [mu] M, and has a complementarity to the D5S818 genetic locus Primers have a final concentration of 1.0-5.0 [mu] M, primers that complementarily bind to the D21S11 genetic locus have a final concentration of 5.0-20.0 [mu] M, primers that bind complementarily to the TPOX genetic locus are 0.1-2.0 [mu] M With a final concentration, the primers complementarily binding to the FGA genetic locus had a final concentration of 0.5-3.0 μM, the primers complementarily binding to the D13S317 genetic locus had a final concentration of 2.0-8.0 μM, the D7S820 genetic loci Primers complementarily binding to the D16S539 genetic locus have a final concentration of 2.0-8.0 μM and primers that complementarily bind to the D19S433 genetic locus Primers that have a final concentration of 0.5-3.0 [mu] M and that bind complementarily to the SE33 genetic locus in the second primer set have a final concentration of 0.1-2.0 [mu] M, The bait-binding primers have a final concentration of 0.1-2.0 μM, the primers that complementarily bind to the D1S1656 genetic locus have a final concentration of 0.1-2.0 μM, the primers that bind complementarily to the Penta D genetic locus are 0.1 With a final concentration of -2.0 [mu] M, the primers that complementarily bind to the D22S1045 genetic locus have a final concentration of 0.1-2.0 [mu] M, the primers that bind complementarily to the D10S1248 genetic locus have a final concentration of 0.5-3.0 [mu] M , Primers complementarily binding to the D12S391 genetic locus have a final concentration of 0.5-3.0 μM, primers that bind complementarily to the D2S441 genetic locus have a final concentration of 0.1-2.0 μM, complementary to the Penta E genetic locus May be characterized by having a final concentration of 0.5-3.0 [mu] M.
본 발명의 멀티플렉스 증폭 산물에서 증폭된 대립유전자를 분리(separate)하기 위한 전기영동(electrophoresis)을 이용하여 실시한다.This is done using electrophoresis to separate the amplified allele in the multiplex amplification product of the present invention.
전기영동은 분산된 공간적으로 동일한 전기장의 영향 하에 입자의 움직임의 흐름에 관한 것이다. 전기영동의 종류로는 친화성 전기영동(Affinity electrophoresis), 모세관 전기영동(Capillary electrophoresis) 및 겔 전기영동(Gel electrophoresis) 등이 있으며, 이에 한정되지 않는다.Electrophoresis relates to the flow of particles under the influence of the same spatially distributed electric field. The types of electrophoresis include, but are not limited to affinity electrophoresis, capillary electrophoresis, and gel electrophoresis.
본 발명의 일 실시예에 따르면, 본 발명의 멀티플렉스 증폭 산물에서 증폭된 대립유전자를 분리하기 위한 모세관 전기영동을 이용하여 실시한다. 상기 모세관 전기영동은 전하를 띄고 있는 이온들에 전기장을 걸어주어 각 이온들은 반대전하를 띤 전극으로 이동하는 데 이때의 이동속도는 각 이온들의 평균 전하, 크기, 모양, 용매의 성질에 따라 달라진다. 모세관 전기영동은 이온들이 가느다란 관을 통하여 존재하도록 한 상태에서 관의 양끝에 전기장을 걸어 줄 때 이온들이 관내에서 그들의 성질에 따라 각기 다른 속도로 일정한 방향성을 가지면서 이동하는 성질을 이용하여 물질을 분리하는 장치이다.According to one embodiment of the present invention, capillary electrophoresis is used to isolate the amplified allele in the multiplex amplification product of the present invention. In the capillary electrophoresis, an electric field is applied to the charged ions, and each of the ions moves to the electrode having the opposite charge. The movement speed depends on the average charge, size, shape, and properties of the solvent. Capillary electrophoresis is a technique that utilizes the property that when ions are applied to both ends of a tube in a state where ions are present through a thin tube, the ions move in the tube with different orientations at different rates according to their properties. It is a device to separate.
본 발명은 다른 관점에서, D8S1179, CSF1PO(Human c-fms protooncogene for CSF-1 receptor gene), D3S1358, D2S1338, 아멜로제닌(Amelogenin), vWA(von Willebrand factor A), TH01, D18S51, D5S818, D21S11, TPOX(Human thyroid peroxidase gene), FGA(Human fibrinogen alpha chain), D13S317, D7S820, D16S539 및 D19S433를 포함하는 유전좌위 각각을 증폭할 수 있는 프라이머를 포함하는 제1프라이머 세트; 및 아멜로제닌, SE33, D1S1656, Penta D, D22S1045, D10S1248, D12S391, D2S441 및 Penta E를 포함하는 유전자 각각을 증폭할 수 있는 프라이머를 포함하는 제2프라이머 세트를 포함하는 인간 객체(human subject) 염색체 상의 STR (short tandem repeat) 분석용 멀티플렉스 유전자 증폭 키트에 관한 것이다.In another aspect of the present invention, there is provided a compound of the present invention, which comprises a D8S1179, a CSF1PO (Human c-fms protooncogene for CSF-1 receptor gene), D3S1358, D2S1338, Amelogenin, vWA, TH01, D18S51, D5S818, , A human thyroid peroxidase gene (TPOX), a human fibrinogen alpha chain (FGA), D13S317, D7S820, D16S539 and D19S433; And a second primer set comprising a primer capable of amplifying each of the genes including amelogenin, SE33, D1S1656, Penta D, D22S1045, D10S1248, D12S391, D2S441 and Penta E, And more particularly to a multiplex gene amplification kit for analyzing STR (short tandem repeat)
본 발명에 있어서, 상기 키트는 반응 완충액, DNA 중합 효소, dNTP(dATP, dCTP, dGTP 및 dTTP를 포함하는 혼합물)를 더 포함할 수 있다. 상기 DNA 중합 효소는 예를 들어, Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis 또는 Pyrococcus furiosus (Pfu)로부터 수득한 열 안정성 DNA 중합 효소일 수 있다. 또한, 반응 완충액은 증폭 반응의 pH를 조절함으로써 증폭 반응의 하나 이상의 구성 요소의 안정성, 활성, 및/또는 수명을 변형시키는 증폭 반응에 첨가되는 화합물로서, 이러한 완충 용액들은 당업계에 잘 알려져 있으며, 예를 들어, Tris, Tricine, MOPS, 또는 HEPES일 수 있으나 이에 한정하지는 않는다. 이 외에도, 상기 키트는 필요에 따라, DNA 중합 효소 조인자를 더 포함할 수 있다.In the present invention, the kit may further comprise a reaction buffer, a DNA polymerase, dNTP (a mixture comprising dATP, dCTP, dGTP and dTTP). The DNA polymerase may be, for example, a thermostable DNA polymerase obtained from Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis or Pyrococcus furiosus (Pfu). Also, the reaction buffer is a compound added to an amplification reaction that modifies the stability, activity, and / or lifetime of one or more components of the amplification reaction by adjusting the pH of the amplification reaction. Such buffer solutions are well known in the art, For example, Tris, Tricine, MOPS, or HEPES. In addition, the kit may further comprise a DNA polymerase joiner, if desired.
실시예Example
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다. Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.
실시예 1. 시료에서 DNA 샘플 수득Example 1. Obtaining a DNA sample from a sample
시료가 모근 또는 구강상피세포일 경우에는 하기의 방법으로 DNA 샘플을 수득하였다(E-Prep DNA extraction solution, Viagen Biotech):When the sample is a hair follicle or oral epithelial cell, a DNA sample was obtained in the following manner (E-Prep DNA extraction solution, Viagen Biotech):
시료에 E-Prep Solution(Viagen Biotech) 100-200μl를 첨가한 다음, Porteinase K(20mg/ml, Sigma Aldrich) 10μl를 첨가한 뒤, 섞어 원심분리를 수행한 다음, 튜브 기벽에 모인 시료를 모은 후 63℃ 항온 수조에서 20분간 배양하고, 다시 85℃ 항온수조에서 15분간 배양한 뒤, 13,000rpm/min으로 4분간 원심 분리하여 상등액 50-100μl를 회수하였다.After adding 100-200 μl of E-Prep Solution (Viagen Biotech) to the sample, 10 μl of Porteinase K (20 mg / ml, Sigma Aldrich) was added and centrifuged to collect the collected sample The cells were cultured in a constant temperature water bath at 63 ° C for 20 minutes and then cultured in a constant temperature water bath at 85 ° C for 15 minutes and then centrifuged at 13,000 rpm / min for 4 minutes to recover 50-100 μl of the supernatant.
시료가 모근, 구강 상피세포가 아닌 경우에는 하기의 방법으로 DNA 샘플을 수득하였다(Qiagen human genomic DNA extraction kit, Qiagen, USA): When the sample is not hair root or oral epithelial cell, a DNA sample was obtained by the following method (Qiagen human genomic DNA extraction kit, Qiagen, USA):
시료에 ATL buffer(Qiagen, USA) 350-400μl를 주입한 다음, 56℃ 항온수조에서 1시간 배양하고, 상등액 전체를 회수하여 다른 튜브에 옮겨 담은 후, AL buffer(Qiagen, USA 350-400μl를 첨가한 다음 70℃ 항온수조에서 10분간 배양한 후, 100% 에탄올 200μl를 첨가하였다. 상기 용액을 column에 넣어 13,000rpm 으로 1분간 원심분리하여 collection tube에 담긴 용액은 버리고 column에 AW1 buffer 650μl를 첨가한 뒤, 13,000rpm 으로 1분간 원심분리한 뒤, collection tube에 담긴 용액을 버린 다음, 상기 과정을 2회 반복하였다. 세척된 column을 1.5ml 튜브에 끼운 다음, 3차 증류수 20μl를 첨가하고 상온에서 5분간 배양한 뒤, 13,000rpm으로 1분간 원심부리하여 최종 genomic DNA를 수득하였다.350-400 μl of ATL buffer (Qiagen, USA) was injected into the sample and incubated for 1 hour in a constant temperature water bath at 56 ° C. The whole supernatant was recovered and transferred to another tube, and then AL buffer (Qiagen, After incubating for 10 minutes in a constant-temperature water bath at 70 ° C, 200 μl of 100% ethanol was added to the column, and the solution was centrifuged at 13,000 rpm for 1 minute to discard the solution in the collection tube and 650 μl of AW1 buffer After centrifugation at 13,000 rpm for 1 minute, the solution in the collection tube was discarded and the procedure was repeated twice. The washed column was inserted into a 1.5 ml tube, and then 20 μl of tertiary distilled water was added thereto. Minute, centrifuged at 13,000 rpm for 1 minute to obtain final genomic DNA.
실시예 2. 듀얼 멀티플렉스 시스템을 이용한 STR 유전좌 증폭Example 2. STR Genetic Left Amplification Using Dual Multiplex System
D18S1179, CSF1PO, D3S1358, D2S1338, Amelogenin, vWA, TH01, D18S51, D5S818, D21S11, TPOX, FGA, D13S317, D7S820, D16S539, D19S433 16개 STR 유전자 좌위들과 SE33, Amelogenin, D1S1656, Penta D, D22S1045, D10S1248, D12S391, D2S441, Penta E의 9개 STR 유전자 좌위들에 각각 대응되는, 형광물질로 표지된 프라이머들을 포함하는 듀얼 멀티플렉스 PCR 시스템을 사용하여 DNA 샘플 내 서열을 PCR 증폭하였다.D21S1179, CSF1PO, D3S1358, D2S1338, Amelogenin, vWA, TH01, D18S51, D5S818, D21S11, TPOX, FGA, D13S317, D7S820, D16S539, D19S433 , D12S391, D2S441, and Penta E, respectively. The sequences in the DNA samples were PCR amplified using a dual multiplex PCR system comprising primers labeled with fluorescent substances, respectively.
PCR 반응물은 20μl를 기준으로 2X PCR master mix(Qiagen, USA) 10μl, 시료 DNA 1-10ng, 10X 프라이머 세트 2μl, DW 나머지의 부피로 제작하였으며, DNA 샘플의 최소 검출 한계치는 0.5~1 ng으로 법의학적 시료들의 특성상 극미량의 검체에 해당하는 프로필도 본 키트를 사용해 검출할 수 있는 민감도를 갖는 것을 확인하였다.The PCR product was prepared in a volume of 20 μl based on a 2 × PCR master mix (Qiagen, USA), 10 μl of sample DNA, 2 μl of 10 × primer set, and DW buffer. The minimum detection limit of DNA samples was 0.5 to 1 ng Due to the nature of the medical samples, it was confirmed that a profile corresponding to a trace amount of the sample had a sensitivity that could be detected using this kit.
각 프라이머 세트별 서열 정보 및 증폭산물 크기는 하기 표 1/2와 같다.Sequence information and amplification product size for each primer set are shown in Table 1 below.
Figure PCTKR2018016847-appb-T000001
Figure PCTKR2018016847-appb-T000001
Figure PCTKR2018016847-appb-T000002
Figure PCTKR2018016847-appb-T000002
듀얼 멀티플렉스 PCR 반응은 하기의 조건으로 수행하였다.Dual multiplex PCR reactions were performed under the following conditions.
Veriti Thermal Cycler (ABI, USA) 혹은 이에 상응하는 성능을 지닌 기기Veriti Thermal Cycler (ABI, USA) or equivalent instrument
(1) 초기반응(predenaturation): 95 ℃ 11분(1) Initial reaction (predenaturation): 95 ° C for 11 minutes
(2) 증폭반응(denaturation, amplification, extension): 30회 사이클링(2) Denaturation, amplification, extension: 30 cycles
95 ℃ 1분 → 60 ℃ 1분 → 72 ℃ 1분95 ℃ for 1 min → 60 ℃ for 1 min → 72 ℃ for 1 min
(3) 최종연장(Final extension): 60 ℃ 60분(3) Final extension: 60 ° C for 60 minutes
(4) 냉각(Chilling): 4 ℃ 지속(4) Chilling: Continuous at 4 ℃
실시예 3. STR 유전좌 결정Example 3. STR Genetic Left Determination
3130XL Genetic Analyzer (ABI, USA), 3730XL Genetic Analyzer (ABI, USA) 및 3500 or 3500XL Genetic Analyzer (ABI, USA)를 이용하여 모세관(capillary) 전기영동을 이용하여 증폭산물의 크기를 결정하였다.Amplification products were determined using capillary electrophoresis using a 3130XL Genetic Analyzer (ABI, USA), a 3730XL Genetic Analyzer (ABI, USA) and a 3500 or 3500XL Genetic Analyzer (ABI, USA).
실시예 2에서 수득한 증폭 산물 1μl와 본 발명에서 제작한 allelic ladder 1μl를 준비한 다음, Hi-Di formamide 9.3μl와 GeneScan-500 LIZ(ABI, USA) 0.05μl를 혼합하고, 상기 증폭 산물 및 래더를 각각 첨가하고 95℃에서 3분간 denaturation 시킨 후, 상기 혼합물을 분석 장비의 모세관에 주입하고, 전기영동에 의해 전개가 완료된 증폭산물을 GeneMapper ID v3.2(ABI, USA) 또는 GeneMApper ID-X(ABI, USA)를 이용하여 래더를 기반으로 크기를 결정하였다.1 μl of the amplification product obtained in Example 2 and 1 μl of the allelic ladder prepared in the present invention were prepared and then mixed with 9.3 μl of Hi-Di formamide and 0.05 μl of GeneScan-500 LIZ (ABI, USA) (ABI, USA) or GeneMApper ID-X (ABI, USA) was added to the capillary of the analytical instrument, and the amplified product was analyzed by electrophoresis. , USA) was used to determine the size based on the ladder.
그 결과 도 2 및 3에 개시된 바와 같이 특정 샘플에 대한 STR 유전좌위를 높은 민감도와 정확도로 검출할 수 있음을 확인하였다.As a result, it was confirmed that the STR genetic loci for a specific sample can be detected with high sensitivity and accuracy, as shown in Figs. 2 and 3.
실시예 4. DNA Allelic Ladder 제작Example 4. Production of DNA Allelic Ladder
본 발명은 검체 DNA에 형광 표지된 PCR 프라이머 세트(중합효소연쇄반응 시발체 세트)를 이용하여 형광이 부착된 검체 DNA 사본을 대량 증폭하고 이렇게 증폭된 DNA를 DNA 염기서열 분석기(DNA sequencer, Genetic analyzer)를 이용하여 분석하는 방법이기 때문에, 증폭된 형광 표지 DNA의 정확한 base-pair의 수를 얻기 위해서는 실험적 오차를 보정해주는 base-pair 표지자(size standard)가 필요하다.The present invention relates to a method for amplifying a large amount of fluorescently labeled sample DNA using fluorescence-labeled PCR primer set (polymerase chain reaction primer set) and then amplifying the amplified DNA with a DNA sequencer (genetic analyzer) , A base-pair marker (size standard) that corrects experimental errors is required to obtain the exact number of base pairs of amplified fluorescent-labeled DNA.
이를 DNA allelic ladder라고 하며 모든 STR 키트에는 각 키트에 적합한 DNA allelic ladder가 필요하며 DNA allelic ladder는 키트의 PCR 프라이머 세트를 이용하여 STR 검색에 사용된 임상검체로부터 homogenous STR을 선별하고, 선별된 STR을 PCR 증폭하여 PCR 증폭산물을 운반체 플라스미드(vector plasmid)에 DNA 클로닝(cloning)하여 allelic ladder library를 제작하였다. Allelic ladder library에서 STR의 각 base-pair 크기에 알맞게 PCR하여 그 산물들을 혼합하여 각 peak balance를 맞추기 위한 농도 조절 과정을 거쳐 제작 완료하였다.This is called DNA allelic ladder. All STR kit requires DNA allelic ladder suitable for each kit. DNA allelic ladder is used to select homogenous STR from clinical specimen used in STR search using PCR primer set of kit, PCR amplification products were cloned into a carrier plasmid (vector plasmid) to prepare an allelic ladder library. The Allelic ladder library was PCR-amplified by PCR to match the size of each base-pair of STR, and the products were mixed to complete the concentration control to adjust each peak balance.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적 기술은 단지 바람직한 실시 양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
본 발명에 따른 인간 객체의 유전자 감식을 위한 분석방법은 인간 유전자 감식에 있어서, 수입제품에 의존도가 100%인 상황에서, 높은 재현성 및 정확도로 유전자 감식이 가능하여 개인식별, 혈족 확인에 있어서 유용하다.The analysis method for gene detection of a human object according to the present invention is useful for identification of individuals and identification of blood vessels because human identification can be performed with high reproducibility and accuracy in a situation where a dependency of imported products is 100% .
전자파일 첨부하였음.I attached an electronic file.

Claims (7)

  1. (a) D8S1179, CSF1PO(Human c-fms protooncogene for CSF-1 receptor gene), D3S1358, D2S1338, 아멜로제닌(Amelogenin), vWA(von Willebrand factor A), TH01, D18S51, D5S818, D21S11, TPOX(Human thyroid peroxidase gene), FGA(Human fibrinogen alpha chain), D13S317, D7S820, D16S539 및 D19S433 포함하는 유전좌위 각각을 증폭할 수 있는 프라이머를 포함하는 제1프라이머 세트; 및 아멜로제닌, SE33, D1S1656, Penta D, D22S1045, D10S1248, D12S391, D2S441 및 Penta E를 포함하는 유전자 각각을 증폭할 수 있는 프라이머를 포함하는 제2프라이머 세트와 인간 객체 DNA 시료를 각각 독립적으로 반응시켜 증폭 시키는 단계; 및(a) D8S1179, CSF1PO (Human c-fms protooncogene for CSF-1 receptor gene), D3S1358, D2S1338, Amelogenin, vWA (von Willebrand factor A), TH01, D18S51, D5S818, D21S11, TPOX thyroid peroxidase gene), a human fibrinogen alpha chain (FGA), D13S317, D7S820, D16S539 and D19S433; And a second primer set comprising primers capable of amplifying each of the genes including amelogenin, SE33, D1S1656, Penta D, D22S1045, D10S1248, D12S391, D2S441 and Penta E and a human primate set ; And
    (b) 상기 단계 (a)의 멀티플렉스 증폭 산물을 이용하여 상기 유전좌위의 대립유전자형을 결정하는 단계;를 포함하는 멀티플렉스 유전자 증폭을 이용한 인간 객체의 유전자 감식을 위한 분석방법.(b) determining an allele of the genetic locus using the multiplex amplification product of step (a). < Desc / Clms Page number 19 >
  2. 제1항에 있어서, 상기 제1프라이머 세트는 서열번호 1 내지 32의 염기서열로 표시되는 것을 특징으로 하는 분석방법.2. The analysis method according to claim 1, wherein the first primer set is represented by the nucleotide sequence of SEQ ID NOS: 1-32.
  3. 제1항에 있어서, 상기 제2프라이머 세트는 서열번호 33 내지 50의 염기서열로 표시되는 것을 특징으로 하는 분석방법.The assay method according to claim 1, wherein the second primer set is represented by the nucleotide sequence of SEQ ID NOS: 33 to 50.
  4. 제1항에 있어서, 상기 단계 (a)의 증폭산물은 80-400bp인 것을 특징으로 하는 분석방법.2. The method according to claim 1, wherein the amplification product of step (a) is 80-400 bp.
  5. 제1항에 있어서, 상기 단계 (b)는 상기 멀티플렉스 증폭 산물에서 증폭된 대립유전자의 크기를 크기 표준물(size standard)과 비교/평가하여 결정하며, 상기 크기 표준물은 DNA 마커 또는 유전좌위-특이적 대립유전자 래더인 것을 특징으로 하는 분석방법.2. The method of claim 1, wherein step (b) comprises determining the size of the amplified allele in the multiplex amplification product by comparing / evaluating the size standard with a size marker, - specific allelic ladder.
  6. 제1항에 있어서, 상기 DNA 시료는 혈액, 정액, 질 세포, 모발, 타액, 소변, 구강세포, 태반세포 또는 태아세포를 포함하는 양수 및 이의 혼합물을 포함하는 군으로부터 선택되는 조직으로부터 분리된 것을 특징으로 하는 방법.The method according to claim 1, wherein the DNA sample is separated from a tissue selected from the group consisting of blood, semen, vaginal cells, hair, saliva, urine, oral cells, amniotic fluid including placental cells or fetal cells, Lt; / RTI >
  7. D8S1179, CSF1PO(Human c-fms protooncogene for CSF-1 receptor gene), D3S1358, D2S1338, 아멜로제닌(Amelogenin), vWA(von Willebrand factor A), TH01, D18S51, D5S818, D21S11, TPOX(Human thyroid peroxidase gene), FGA(Human fibrinogen alpha chain), D13S317, D7S820, D16S539 및 D19S433를 포함하는 유전좌위 각각을 증폭할 수 있는 프라이머를 포함하는 제1프라이머 세트; 및 아멜로제닌, SE33, D1S1656, Penta D, D22S1045, D10S1248, D12S391, D2S441 및 Penta E를 포함하는 유전자 각각을 증폭할 수 있는 프라이머를 포함하는 제2프라이머 세트를 포함하는 인간 객체(human subject) 염색체 상의 STR (short tandem repeat) 분석용 멀티플렉스 유전자 증폭 키트.D3S1358, D2S1338, Amelogenin, vW (von Willebrand factor A), TH01, D18S51, D5S818, D21S11, TPOX (Human thyroid peroxidase gene ), A first primer set comprising a primer capable of amplifying each of the genetic loci including human fibrinogen alpha chain (FGA), D13S317, D7S820, D16S539 and D19S433; And a second primer set comprising a primer capable of amplifying each of the genes including amelogenin, SE33, D1S1656, Penta D, D22S1045, D10S1248, D12S391, D2S441 and Penta E, Multiplex gene amplification kit for STR (short tandem repeat) analysis.
PCT/KR2018/016847 2017-12-28 2018-12-28 Method for analyzing str of human subject by using multiplex system, and analysis kit using same WO2019132582A1 (en)

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* Cited by examiner, † Cited by third party
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US20100099082A1 (en) * 2006-06-01 2010-04-22 St. Anna Kinderkrebsforschung Allele Detection
KR101008828B1 (en) * 2010-03-12 2011-01-19 대한민국 Multiplex pcr system comprising 16 str loci and amelogenin which are highly discriminative in korean population and the method of human identification using them
JP2013513379A (en) * 2009-12-11 2013-04-22 ニュークレイックス Classification of DNA samples
KR101457983B1 (en) * 2014-05-15 2014-11-06 대한민국 Method for Autosomal Analysing Human Subject of Analytes Using Multiplex Gene Amplification
KR101667526B1 (en) * 2015-12-30 2016-10-19 대한민국 Method for Extended Autosomal STR Analysing Human Subject of Analytes using a Next Generation Sequencing Technology

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100099082A1 (en) * 2006-06-01 2010-04-22 St. Anna Kinderkrebsforschung Allele Detection
JP2013513379A (en) * 2009-12-11 2013-04-22 ニュークレイックス Classification of DNA samples
KR101008828B1 (en) * 2010-03-12 2011-01-19 대한민국 Multiplex pcr system comprising 16 str loci and amelogenin which are highly discriminative in korean population and the method of human identification using them
KR101457983B1 (en) * 2014-05-15 2014-11-06 대한민국 Method for Autosomal Analysing Human Subject of Analytes Using Multiplex Gene Amplification
KR101667526B1 (en) * 2015-12-30 2016-10-19 대한민국 Method for Extended Autosomal STR Analysing Human Subject of Analytes using a Next Generation Sequencing Technology

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