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KR-102961489-B1 - DNA Typing Kits using Minisatellites Polymorphism

KR102961489B1KR 102961489 B1KR102961489 B1KR 102961489B1KR-102961489-B1

Abstract

The present invention relates to a DNA typing kit utilizing the polymorphic properties of genes. It was confirmed that the polymorphic markers of the present invention— MUC2 -MS3, MUC2 -MS4, MUC2 -MS8, MUC4 - MS5, MUC5B - MS6, MUC8 -MS5, BORIS-MS2, SHC2 - MS1, SHC2 -MS2, SHC2 -MS3, SLC6A18 -MS1, SLC6A18-MS2, SLC6A18 -MS6, and SLC6A19 -MS1—can be used as markers for individual identification by confirming that the number of repeats differs between individuals. Furthermore, it was confirmed that the above markers are transmitted through meiosis according to Mendelian inheritance, and that by DNA typing, they can be effectively used for paternity testing, kinship verification, or forensic analysis of individuals.

Inventors

  • 임선희
  • 문정연
  • 정미소
  • 김민혜
  • 양기은
  • 정민영

Assignees

  • 주식회사 메딕바이오엔케이
  • 소프텍코리아주식회사

Dates

Publication Date
20260507
Application Date
20220318

Claims (14)

  1. A polymorphic minisatellite marker composition for personal identification comprising: MUC4 -MS5 ( MUC4 -minisatellite 5), a polymorphic minisatellite in the intron 5 region of the MUC4 gene containing the nucleotide sequence of SEQ ID NO. 4; SHC2 -MS1 ( SHC2 -minisatellite 1), a polymorphic minisatellite in the intron 10 region of the SHC2 gene containing the nucleotide sequence of SEQ ID NO. 8 ; SHC2 -MS2 ( SHC2 -minisatellite 2), a polymorphic minisatellite in the intron 9 region of the SHC2 gene containing the nucleotide sequence of SEQ ID NO. 9; and SHC2 -MS3 ( SHC2 -minisatellite 3), a polymorphic minisatellite in the intron 9 region of the SHC2 gene containing the nucleotide sequence of SEQ ID NO. 10.
  2. In Article 1, The above composition comprises: a polymorphic subspecies MUC2 -MS3 ( MUC2 -minisatellite 3) of the intron 20 region of the MUC2 gene containing the nucleotide sequence of SEQ ID NO. 1; a polymorphic subspecies MUC2-MS4 (MUC2-minisatellite 4) of the intron 23 region of the MUC2 gene containing the nucleotide sequence of SEQ ID NO. 2; a polymorphic subspecies MUC2 -MS8 ( MUC2 -minisatellite 8) of the intron 43 region of the MUC2 gene containing the nucleotide sequence of SEQ ID NO. 3; and a polymorphic subspecies MUC5B - MS6 ( MUC5B -minisatellite 6) of the exon 37 and intron 37 regions of the MUC5B gene containing the nucleotide sequence of SEQ ID NO. 5. The polymorphic subspecies MUC8 -MS5 ( MUC8 -minisatellite 5) of the intron 1 and exon 2 regions of the MUC8 gene containing the nucleotide sequence of SEQ ID NO. 6; the polymorphic subspecies BORIS -MS2 ( BORIS -minisatellite 2) of the promoter region of the BORIS gene containing the nucleotide sequence of SEQ ID NO. 7; the polymorphic subspecies SLC6A18 - MS1 ( SLC6A18 -minisatellite 1) of the promoter region of the SLC6A18 gene containing the nucleotide sequence of SEQ ID NO. 11; the polymorphic subspecies SLC6A18 -MS2 ( SLC6A18 -minisatellite 2) of the intron 1 region of the SLC6A18 gene containing the nucleotide sequence of SEQ ID NO. 12; A composition further comprising a polymorphic subspecies consisting of: SLC6A18 -MS6 ( SLC6A18 -minisatellite 6), a polymorphic subspecies of the intron 7 region of the SLC6A18 gene containing the nucleotide sequence of SEQ ID NO. 13; and SLC6A19 -MS1 ( SLC6A19 -minisatellite 1), a polymorphic subspecies of the promoter region of the SLC6A19 gene containing the nucleotide sequence of SEQ ID NO. 14.
  3. In Article 1, The above marker is a composition that is inherited through meiosis.
  4. A primer set for detecting polymorphic minisatellites composed of MUC4-MS5 , SHC2-MS1, SHC2-MS2, and SHC2-MS3, comprising a primer set consisting of SEQ ID NOs 21 and 22; a primer set consisting of SEQ ID NOs 29 and 30; a primer set consisting of SEQ ID NOs 31 and 32; and a primer set consisting of SEQ ID NOs 33 and 34.
  5. In Paragraph 4, The above sequence numbers 21 and 22 amplify polymorphic so-called MUC4 -MS5; The above sequence numbers 29 and 30 amplify polymorphic so-called SHC2 -MS1; The above sequence numbers 31 and 32 amplify polymorphic so-called SHC2 -MS2; The above sequence numbers 33 and 34 are primer sets for amplifying polymorphic so-called SHC2 -MS3.
  6. In Paragraph 4, A primer set comprising a polymorphic so - called ​
  7. In Paragraph 4, The primer set further comprises: a primer set consisting of SEQ ID NOs 15 and 16; a primer set consisting of SEQ ID NOs 17 and 18; a primer set consisting of SEQ ID NOs 19 and 20; a primer set consisting of SEQ ID NOs 23 and 24; a primer set consisting of SEQ ID NOs 25 and 26; a primer set consisting of SEQ ID NOs 27 and 28; a primer set consisting of SEQ ID NOs 35 and 36; a primer set consisting of SEQ ID NOs 37 and 38; a primer set consisting of SEQ ID NOs 39 and 40; and a primer set consisting of SEQ ID NOs 41 and 42.
  8. In Article 7, The above sequence numbers 15 and 16 amplify polymorphic so-called MUC2 -MS3; The above sequence numbers 17 and 18 amplify the polymorphic so-called MUC2 -MS4; The above sequence numbers 19 and 20 amplify the polymorphic so-called MUC2 -MS8; The above sequence numbers 23 and 24 amplify the polymorphic so-called MUC5B -MS6; The above sequence numbers 25 and 26 amplify the polymorphic so-called MUC8 -MS5; The above sequence numbers 27 and 28 amplify the polymorphic so-called BORIS -MS2; The above sequence numbers 35 and 36 amplify the polymorphic so-called SLC6A18 -MS1; The above sequence numbers 37 and 38 amplify the polymorphic so-called SLC6A18 -MS2; The above sequence numbers 39 and 40 amplify the polymorphic so-called SLC6A18 -MS6; The above sequence numbers 41 and 42 are primer sets for amplifying polymorphic so-called SLC6A19 -MS1.
  9. A DNA typing kit for the detection of polymorphic minisatellites comprising MUC4 -MS5, SHC2 -MS1, SHC2 -MS2, and SHC2 -MS3, comprising the primer set of claim 4.
  10. In Article 9, A DNA typing kit comprising the above polymorphic subtype further including polymorphic subtypes consisting of MUC2 -MS3, MUC2 -MS4, MUC2 -MS8, MUC5B -MS6, MUC8 -MS5, BORIS -MS2, SLC6A18 -MS1, SLC6A18 -MS2, SLC6A18 -MS6 and SLC6A19 -MS1.
  11. (a) a step of performing a DNA polymerase chain reaction (PCR) using the kit of claim 9 and genomic DNA extracted from a sample as a template; and (b) A DNA typing method comprising the step of separating the PCR product by electrophoresis to identify polymorphic minisatellites consisting of MUC4 -MS5, SHC2 -MS1, SHC2 -MS2 and SHC2 -MS3.
  12. In Paragraph 11, A method in which the above polymorphic subspecies further comprises polymorphic subspecies consisting of MUC2 -MS3, MUC2 -MS4, MUC2 -MS8, MUC5B -MS6, MUC8 -MS5, BORIS -MS2, SLC6A18 -MS1, SLC6A18 -MS2, SLC6A18 -MS6 and SLC6A19 -MS1.
  13. In Paragraph 11 A method in which the above specimen is selected from the group consisting of blood, oral epithelial cells, hair, saliva, epidermis, semen, vaginal collections, isolated cells, tissue samples, dandruff, and remains.
  14. In claim 11, the above DNA typing method is characterized by being used for paternity testing, blood relationship testing, or forensic examination of an individual.

Description

DNA Typing Kits using Minisatellites Polymorphism The present invention relates to a DNA typing kit utilizing the so-called polymorphism of genes. Among various repetitive sequences, tandem repeat (TR) sequences account for more than 10% of the human genome. Minisatellites (MS) belonging to this category are composed of repeating units with lengths ranging from 10 to 100 bp, and their lengths vary significantly from several hundred bp to over 20 kb depending on the number of repetitions. TR sequences are classified into monomorphic, in which only one allele appears with the same number of repetitions in all individuals, and polymorphic, in which two or more alleles exhibit different repetition counts and appear in different patterns for each person. Since the lengths of these repetitive sequences are stably transmitted from parents to offspring through meiosis, they can be used for paternity testing or as forensic markers. Accordingly, the inventors completed the present invention by confirming that the DNA typing markers can be used for personal identification through TR analysis and polymorphism verification within the MUC2 , MUC4 , MUC5B , MUC8 , BORIS , SHC2 , SLC6A18 , and SLC6A19 genes. Figure 1 shows the analysis of polymorphisms of MUC2 -MS3 amplified from genomic DNA isolated from the blood and oral epithelial cells of an individual by electrophoresis (A: blood genomic DNA analysis, B: oral epithelial cell genomic DNA analysis). Figure 2 shows the analysis of polymorphisms of MUC2 -MS4 amplified from genomic DNA isolated from the blood and oral epithelial cells of an individual by electrophoresis (A: blood genomic DNA analysis, B: oral epithelial cell genomic DNA analysis). Figure 3 shows the polymorphism of MUC2 -MS8 amplified from genomic DNA isolated from the blood and oral epithelial cells of an individual, analyzed by electrophoresis (A: blood genomic DNA analysis, B: oral epithelial cell genomic DNA analysis). Figure 4 shows the analysis of the polymorphism of MUC4 -MS5 amplified from genomic DNA isolated from the blood and oral epithelial cells of an individual by electrophoresis (A: blood genomic DNA analysis, B: oral epithelial cell genomic DNA analysis). Figure 5 shows the analysis of the polymorphism of MUC5B -MS6 amplified from genomic DNA isolated from the blood and oral epithelial cells of an individual by electrophoresis (A: blood genomic DNA analysis, B: oral epithelial cell genomic DNA analysis). Figure 6 shows the analysis of the polymorphism of MUC8 -MS5 amplified from genomic DNA isolated from the blood and oral epithelial cells of an individual by electrophoresis (A: blood genomic DNA analysis, B: oral epithelial cell genomic DNA analysis). Figure 7 shows the polymorphism of BORIS -MS2 amplified from genomic DNA isolated from individual blood and oral epithelial cells, analyzed by electrophoresis (A: blood genomic DNA analysis, B: oral epithelial cell genomic DNA analysis). Figure 8 shows the polymorphism of SHC2 -MS1 amplified from genomic DNA isolated from the blood and oral epithelial cells of an individual, analyzed by electrophoresis (A: blood genomic DNA analysis, B: oral epithelial cell genomic DNA analysis). Figure 9 shows the polymorphism of SHC2 -MS2 amplified from genomic DNA isolated from individual blood and oral epithelial cells, analyzed by electrophoresis (A: blood genomic DNA analysis, B: oral epithelial cell genomic DNA analysis). Figure 10 shows the analysis of polymorphisms of SHC2 -MS3 amplified from genomic DNA isolated from the blood and oral epithelial cells of an individual by electrophoresis (A: blood genomic DNA analysis, B: oral epithelial cell genomic DNA analysis). Figure 11 shows the polymorphism of SLC6A18 -MS1 amplified from genomic DNA isolated from individual blood and oral epithelial cells, analyzed by electrophoresis (A: blood genomic DNA analysis, B: oral epithelial cell genomic DNA analysis). Figure 12 shows the polymorphism of SLC6A18 -MS2 amplified from genomic DNA isolated from individual blood and oral epithelial cells, analyzed by electrophoresis (A: blood genomic DNA analysis, B: oral epithelial cell genomic DNA analysis). Figure 13 shows the polymorphism of SLC6A18 -MS6 amplified from genomic DNA isolated from the blood and oral epithelial cells of an individual, analyzed by electrophoresis (A: blood genomic DNA analysis, B: oral epithelial cell genomic DNA analysis). Figure 14 shows the polymorphism of SLC6A19 -MS1 amplified from genomic DNA isolated from individual blood and oral epithelial cells, analyzed by electrophoresis (A: blood genomic DNA analysis, B: oral epithelial cell genomic DNA analysis). Figure 15 is a figure confirming the genetic transfer of the polymorphic minisatellite (MS) of the present invention through meiosis. Embodiments of the present invention will be described in detail below with reference to the attached drawings. In the following description, detailed descriptions of technologies well known to those