Method and kit for detection of mutations in mitochondrial dna

• US 20060078881A1
• Filed: 03/13/2003
• Published: 04/13/2006
• Est. Priority Date: 03/15/2002
• Status: Abandoned Application

• Current Assignee: Hanna Andreasson, Marie Allen, Ulf Gyllensten
• Original Assignee: Hanna Andreasson, Marie Allen, Ulf Gyllensten
• Inventors: Gyllensten, Ulf, Andreasson, Hanna, Allen, Marie
• Application Number:
  US10/507,873
• Publication Number:
  US 20060078881A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

The present invention is within the medical field. More precisely, the invention relates to a method and kit for defection of mutations/polymorphisms in human mitochondrial DNA sequences and specifically to the use of mitochondrial DNA variants (polymorphisms) with high mutation frequency to be employed in the comparison of biological samples with samples of known origin in the purpose of, for example, human identification or forensic genetics.

Claims

1. A method for detection of mutations/polymorphisms in a sample of human mitochondrial DNA comprising the following steps:

(a) determining the presence or absence of polymorphic sites having a frequency of mutation of less than 3% in the general population but at least 3% in the Caucasian population according to Table 1 in the nucleic acid sequence of the mitochondrial genome in said sample from a human subject; and

(b) relating the information from step (a) to mitochondrial nucleic acid sequence information of known origin; and

(c) relating the information from step (a), where in one or more of the mitochondrial fragments 1, 4, 12, 14, 15, 16, 19, 20, 24, 25, 26, and 27 in Table 1, to be used for determination of polymorphic site(s).

2. A method according to claim 1, wherein the frequency of mutations is at least 5%.

3. A method according to claim 1, wherein the known information in step (b) is derived from a database of nucleic acid sequence information from humans of diverse origin.

4. A method according to claim 1, wherein the polymorphic sites are detected by assays such as DNA hybridization assays (ASO, SSO hybridization, DNA microchip, padlock), enzymatic ligation assays (OLA, padlock), enzymatic cleavage assays (EMD, Taqman), enzymatic extension assays (mini-sequencing) or other assays for typing of genetic polymorphisms.

5. A method according to claim 1, wherein the mitochondrial polymorphic sites(s) is/are determined by sequencing.

6. A method according to claim 5, wherein the sequencing method is sequencing-by-synthesis.

7. A method according to claim 5, wherein the sequencing method is pyrosequencing.

8. A method according to claim 1, using the primers listed in Table 2.

17. A method according to claim 1, wherein the frequency of mutations is at least 10%.

18. A method according to claim 1, wherein the frequency of mutations is at least 15%.

9. A kit for detecting the detecting mutations/polymorphism in the human mtDNA, comprising means for analysis of the polymorphic sites having a frequency of mutation of at least 3% according to Table 1.

10. A method according to claim 9, comprising means for analysis of the polymorphic sites having a frequency of mutation of at least 5% according to Table 1.

11. A kit according to claim 9, comprising one or more of the sequencing primers in Table 2.

12. A kit according to claim 9, comprising two or more amplification primers according to Table 2 for fragment 1, 4, 12, 14, 15, 16, 19, 20, 24, 25, 26, and 27 according to Table 1.

13. A kit according to claim 9, wherein the means for analysis are sequencing-by-synthesis reagents.

14. A kit according to claim 9, wherein the means for analysis are sequencing reagents.

15. A kit according to claim 9, wherein the means for analysis are pyrosequencing reagents.

16. A kit according to claim 11, wherein two or more of the sequencing primers in Table 2 are attached to a solid support, such as a microtierplate well or array.

19. A kit according to claim 9, comprising means for analysis of the polymorphic sites having a frequency of mutation of at least 10% according to Table 1.

20. A kit according to claim 9, comprising means for analysis of the polymorphic sites having a frequency of mutation of at least 15% according to Table 1.