OLIGONUCLEOTIDE PRIMERS FOR QUANTIFYING AND/OR DETECTING HUMAN MALE DNA AND KITS CONTAINING THE SAME

Abstract

Oligonucleotide primers and probes for quantifying and/or detecting human male DNA in a forensic sample. The oligonucleotides are useful for amplifying a multicopy locus within the human Y-chromosome (MCL-Y) that shares at least 80% sequence identity to a sequence according to SEQ ID NO. 3 over a stretch of at least 60 base pairs. The oligonucleotides hybridize under stringent conditions to a nucleic acid having at least one sequence selected from the group consisting of SEQ ID NO. 3 to SEQ ID NO. 11 and/or SEQ ID NO. 17 to SEQ ID NO. 25. Kits including the oligonucleotide primers, probes and/or primer pairs and reagents for performing an amplification reaction on DNA recovered from a forensic sample are also disclosed.

Claims

1. An oligonucleotide primer or probe that hybridizes under stringent conditions to a nucleic acid having at least one sequence selected from the group consisting of: SEQ ID NO. 3; SEQ ID NO. 4; SEQ ID NO. 5; SEQ ID NO. 6; SEQ ID NO. 7; SEQ ID NO. 8; SEQ ID NO. 9; SEQ ID NO. 10; SEQ ID NO. 11; SEQ ID NO. 17; SEQ ID NO. 18; SEQ ID NO. 19; SEQ ID NO. 20; SEQ ID NO. 21; SEQ ID NO. 22; SEQ ID NO. 23; SEQ ID NO. 24; and SEQ ID NO. 25.

2. The oligonucleotide primer or probe according to claim 1, wherein the oligonucleotide primer or probe is selected from the group consisting of: SEQ ID NO. 1; SEQ ID NO. 2; SEQ ID NO. 12; SEQ ID NO. 13; SEQ ID NO. 14; SEQ ID NO. 15; and SEQ ID NO. 16.

3. The oligonucleotide primer or probe according to claim 1, wherein the oligonucleotide primer or probe is selected from the group consisting of: the reverse complement of SEQ ID NO. 1; the reverse complement of SEQ ID NO. 2; the reverse complement of SEQ ID NO. 12; the reverse complement of SEQ ID NO. 13; the reverse complement of SEQ ID NO. 14; the reverse complement of SEQ ID NO. 15; and the reverse complement of SEQ ID NO. 16.

4. The oligonucleotide primer or probe according to claim 1, wherein the oligonucleotide primer or probe is selected from the group consisting of: a primer that shares at least 90% sequence identity with SEQ ID NO. 1; a primer that shares at least 90% sequence identity with SEQ ID NO. 2; a probe that shares at least 90% sequence identity with SEQ ID NO. 12; a primer that shares at least 90% sequence identity with SEQ ID NO. 13; a primer that shares at least 90% sequence identity with SEQ ID NO. 14; a primer that shares at least 90% sequence identity with SEQ ID NO. 15; and a probe that shares at least 90% sequence identity with SEQ ID NO. 16.

5. The oligonucleotide primer according to claim 1, wherein the oligonucleotide primer or probe is selected from the group consisting of: the reverse complement of a primer that shares at least 90% sequence identity with SEQ ID NO. 1; the reverse complement of a primer that shares at least 90% sequence identity with SEQ ID NO. 2; the reverse complement of a primer that shares at least 90% sequence identity with SEQ ID NO. 12; the reverse complement of a primer that shares at least 90% sequence identity with SEQ ID NO. 13; the reverse complement of a primer that shares at least 90% sequence identity with SEQ ID NO. 14; the reverse complement of a primer that shares at least 90% sequence identity with SEQ ID NO. 15; and the reverse complement of a primer that shares at least 90% sequence identity with SEQ ID NO. 16.

6. An oligonucleotide primer pair selected from the group consisting of: SEQ ID NO. 1 and SEQ ID NO. 2; SEQ ID NO. 2 and SEQ ID NO. 13; SEQ ID NO. 2 and SEQ ID NO. 14; SEQ ID NO. 2 and SEQ ID NO. 15; the reverse complement of SEQ ID NO. 1 and the reverse complement of SEQ ID NO. 2; the reverse complement of SEQ ID NO. 2 and the reverse complement of SEQ ID NO. 13; the reverse complement of SEQ ID NO. 2 and the reverse complement of SEQ ID NO. 14; the reverse complement of SEQ ID NO. 2 and the reverse complement of SEQ ID NO. 15; a primer that shares at least 90% sequence identity with SEQ ID NO. 1 and a primer that shares at least 90% sequence identity with SEQ ID NO. 2; a primer that shares at least 90% sequence identity with SEQ ID NO. 2 and a primer that shares at least 90% sequence identity with SEQ ID NO. 13; a primer that shares at least 90% sequence identity with SEQ ID NO. 2 and a primer that shares at least 90% sequence identity with SEQ ID NO. 14; a primer that shares at least 90% sequence identity with SEQ ID NO. 2 and a primer that shares at least 90% sequence identity with SEQ ID NO. 15; the reverse complement of a primer that shares at least 90% sequence identity with SEQ ID NO. 1 and the reverse complement of a primer that shares at least 90% sequence identity with SEQ ID NO. 2; the reverse complement of a primer that shares at least 90% sequence identity with SEQ ID NO. 2 and the reverse complement of a primer that shares at least 90% sequence identity with SEQ ID NO. 13; the reverse complement of a primer that shares at least 90% sequence identity with SEQ ID NO. 2 and the reverse complement of a primer that shares at least 90% sequence identity with SEQ ID NO. 14; and the reverse complement of a primer that shares at least 90% sequence identity with SEQ ID NO. 2 and the reverse complement of a primer that shares at least 90% sequence identity with SEQ ID NO. 15.

7. The oligonucleotide primer pair according to claim 6, wherein the primer pair is a primer that shares at least 90% sequence identity with SEQ ID NO. 1 and a primer that shares at least 90% sequence identity with SEQ ID NO. 2, or the reverse complements thereof.

8. The oligonucleotide primer pair according to claim 6, wherein the primer pair is a primer that shares at least 90% sequence identity with SEQ ID NO. 2 and a primer that shares at least 90% sequence identity with SEQ ID NO. 13, or the reverse complements thereof.

9. The oligonucleotide primer pair according to claim 6, wherein the primer pair is a primer that shares at least 90% sequence identity with SEQ ID NO. 2 and a primer that shares at least 90% sequence identity with SEQ ID NO. 14, or the reverse complements thereof.

10. The oligonucleotide primer pair according to claim 6, wherein the primer pair is a primer that shares at least 90% sequence identity with SEQ ID NO. 2 and a primer that shares at least 90% sequence identity with SEQ ID NO. 15, or the reverse complements thereof.

11. A kit comprising an oligonucleotide primer or probe according to claim 1 and reagents for performing an amplification reaction on DNA recovered from a forensic sample.

12. A kit comprising an oligonucleotide primer or probe according to claim 2 and reagents for performing an amplification reaction on DNA recovered from a forensic sample.

13. A kit comprising an oligonucleotide primer or probe according to claim 3 and reagents for performing an amplification reaction on DNA recovered from a forensic sample.

14. A kit comprising an oligonucleotide primer or probe according to claim 4 and reagents for performing an amplification reaction on DNA recovered from a forensic sample.

15. A kit comprising an oligonucleotide primer or probe according to claim 5 and reagents for performing an amplification reaction on DNA recovered from a forensic sample.

16. A kit comprising an oligonucleotide primer pair according to claim 6 and reagents for performing an amplification reaction on DNA recovered from a forensic sample.

17. A kit comprising an oligonucleotide primer pair according to claim 7 and reagents for performing an amplification reaction on DNA recovered from a forensic sample.

18. A kit comprising an oligonucleotide primer pair according to claim 8 and reagents for performing an amplification reaction on DNA recovered from a forensic sample.

19. A kit comprising an oligonucleotide primer pair according to claim 9 and reagents for performing an amplification reaction on DNA recovered from a forensic sample.

20. A kit comprising an oligonucleotide primer pair according to claim 10 and reagents for performing an amplification reaction on DNA recovered from a forensic sample.

Description

FIGURES

[0082] FIG. 1 shows the superiority of the present method (Investigator Quantiplex Pro) compared to other methods available on the market due to its increased sensitivity.

[0083] FIG. 2 shows a possible amplification set-up. It shows different combinations of primer pairs for the amplification of a multicopy locus within the Y-chromosome (MCL-Y).

[0084] FIG. 3 Measurement of degraded male DNA according to the invention in humans.

[0085] The invention shows no significant increase for the Ct values for the smallest PCR system (81 bp) for compromised DNA with an average fragment length from 1500 bp and 500 bp. Only for 300 bp and 150 bp there is an increase of Ct values. Surprisingly the larger PCR system (359 bp) shows already a significant shift of Ct values when applied on fragmented DNA of 1500 bp length. Furthermore, the Ct values increase consistently on every further tested fragment length from 500 bp, 300 bp, to 150 bp and reach their maximum at 150 bp with more than 8 Ct values compared to undegraded DNA. This allows for a precise assessment of the degradation or integrity status of male DNA. 2.3 ng/μl of male DNA was used for every fragment size or undegraded male DNA.

[0086] FIG. 4 Degradation index generated by the invention.

[0087] Shown are the degradation indices (i.e. the ratio of the amount of short fragments vs. the amount of long fragments (male S/male L)) of the invention. Noticeably, the method according to the invention (second column) obtains extremely high indices, in particular for the small fragments (a value of almost 190, when 2.3 ng/μl of male DNA was tested). This indicates a high sensitivity for the detection of degraded male DNA.

[0088] FIG. 5 Measurement of degraded male DNA in background of female DNA in humans.

[0089] Different fragmented male DNAs (each 0.76 ng/μl) have been spiked into non degraded female DNA (32 ng/μl). The invention shows no significant increase for the Ct values for the smallest PCR system (81 bp) for compromised DNA with an average fragment length for 500 bp. Only for 300 bp and 150 bp there is an increase of Ct values. Surprisingly the larger PCR system (359 bp) shows a significant shift of Ct values when applied on fragmented DNA of 500 bp length. Furthermore, the Ct values increase consistently on every further tested fragment length from 300 bp, to 150 bp and reach their maximum at 150 bp with more than 8 Ct values compared to undegraded DNA. This allows for a precise assessment of the degradation or integrity status of male DNA in female background DNA.

[0090] FIG. 6 Detection of male cell-free fetal DNA in cell-free DNA from pregnant women.

[0091] Shown is the Degradation Index (DI) generated by applying the invention on isolated cell free DNA from a pregnant woman. The system is able to detect low amounts of male DNA with both PCR systems for male targets, the small (81 bp) and the large one (359 bp). The small system detects the male cell free fetal DNA which is sized between 150-220 bp. The large male PCR system performs similarly to the small PCR system only on pure male genomic DNA or on contaminating male genomic DNA (spike in controls); on male cell free fetal DNA the performance will significantly drop due to the size limitation of the fragments of male cell-free fetal DNA in the cell-free DNA from pregnant women. This generates a high Degradation Index (DI) for non-contaminated cell-free DNA from women pregnant with a male embryo and a lower degradation index for contaminated cell-free DNA from women pregnant with a male or female embryo.