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EP-3516072-B1 - METHOD FOR QUANTIFYING AND/OR DETECTING HUMAN MALE DNA

EP3516072B1EP 3516072 B1EP3516072 B1EP 3516072B1EP-3516072-B1

Inventors

  • VRANES, Miroslav
  • PEIST, RALF
  • SCHERER, MARIO
  • CORNELIUS, Stefan Otto
  • KÖNIG, Margaretha

Dates

Publication Date
20260506
Application Date
20170915

Claims (14)

  1. A method for detecting, assessing the status of and/or quantifying DNA, in particular the fraction of male DNA, in a sample, wherein the method comprises the step of a. amplifying a multicopy locus within the Y-chromosome (MCL-Y), wherein the multicopy locus shares at least 85% sequence identity to a sequence according to SEQ ID NO. 3 over a stretch of at least 60 base pairs (bp) or with the reverse complement thereof or, wherein the multicopy locus is amplifiable with a primer pair according to SEQ ID NO. 1 and SEQ ID NO. 2 or the reverse complement thereof, wherein the amplification reaction comprises amplifying at least two overlapping regions using at least one common primer, wherein the step of amplifying a multicopy locus within the Y-chromosome (MCL-Y) generates at least two fragments, comprising a short fragment and a large fragment, wherein the method further comprises the step of measuring a ratio between the short fragment over the large fragment, thereby obtaining a degradation index which indicates the status of and/or quantifies DNA.
  2. The method according to claim 1, wherein the amplification step is performed using at least one primer selected from one of the groups consisting of: a. SEQ ID NO.1 and SEQ ID NO. 2, b. a reverse complement of SEQ ID NO. 1 and SEQ ID NO. 2 and c. a primer that shares at least 90% sequence identity with one of the primers with SEQ ID NO. 1 and SEQ ID NO. 2 or a reverse complement thereof.
  3. The method according to any of the claims 1 or 2, wherein the amplification step is performed using a primer pair selected from one of the groups consisting of: a. SEQ ID NO.1 and SEQ ID NO. 2, b. a reverse complement of SEQ ID NO. 1 and SEQ ID NO. 2, and c. a primer that shares at least 90% sequence identity with one of the primers with SEQ ID NO. 1 and SEQ ID NO. 2 or a reverse complement thereof.
  4. The method according to any of the claims 1 to 3, wherein the amplification is performed using a primer pair with a sequence according to SEQ ID NO. 1 and SEQ ID NO. 2, in combination with at least one primer pair selected from the group consisting of: a. SEQ ID NO. 2 and SEQ ID NO. 13 and/or SEQ ID NO. 2 and SEQ ID NO. 14 and/or SEQ ID NO. 2 and SEQ ID NO. 15, b. a reverse complement of SEQ ID NO. 2 and SEQ ID NO. 13 and/or SEQ ID NO. 2 and SEQ ID NO. 14 and/or SEQ ID NO. 2 and SEQ ID NO. 15, c. a primer that shares at least 90% sequence identity with one of the primers with SEQ ID NO. 2 and SEQ ID NO. 13 and/or SEQ ID NO. 2 and SEQ ID NO. 14 and/or SEQ ID NO. 2 and SEQ ID NO. 15.
  5. The method according to any of the claims 1-4, wherein the amplification reaction using primers according to SEQ ID NO. 1 and SEQ ID NO. 2 is detected by a probe according to SEQ ID NO. 12 and/or, wherein the products of the larger amplification reaction using primers according to SEQ ID NO. 2 and SEQ ID NO. 13 and/or SEQ ID NO. 2 and SEQ ID NO. 14 and/or SEQ ID NO. 2 and SEQ ID NO. 15 are detected by a probe according to SEQ ID NO. 16.
  6. The method according to any of the claims 1 to 5, wherein said sample originates from one of the following tissue types comprising whole blood, blood fractions, oral specimen, urine, human bioptic tissue or other parts of the human body upon availability for isolation of a genome.
  7. The method according to any of the claims 1 to 6, wherein said sample comprises male and female genomic DNA.
  8. The method according to any of the claims 1 to 7, wherein said sample also comprises non-degraded or degraded female DNA.
  9. The method according to any of the claims 1 to 8, wherein the amplification method is a polymerase chain reaction (PCR) or a real-time PCR reaction and the amount of nucleic acid determined is quantified either during the amplification process or as an end point measurement at the end of the amplification reaction.
  10. The method according to any of the claims 1 to 9, wherein the amplification reaction comprises: a. Tris-HCl at a pH of between 8 and 8.8 and/or, b. potassium salt selected from the group of, potassium chloride and potassium sulphate and/or, c. an ammonium salt, preferably ammonium chloride or ammonium sulphate and/or, d. magnesium chloride and/or, e. a hot-start polymerase.
  11. Use of a probe according to SEQ ID NO. 12 or SEQ ID NO. 16 for the detection of the amplification reaction in a method according to claim 1.
  12. Use of an oligonucleotide primer or primer pair, wherein at least one primer, preferably both primers, of said primer pair hybridizes under stringent conditions to a nucleic acid with a sequence according to SEQ ID NO. 3 to SEQ ID NO. 11 in a method according to claim 1.
  13. Kit for performing a method according to any of the claims 1 to 10, wherein said kit comprises at least one oligonucleotide primer according to SEQ ID NO. 2, and a probe according to SEQ ID NO. 12 and/or SEQ ID NO. 16.
  14. Use of oligonucleotides with the following sequences 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 or oligonucleotides that share a sequence identity of no less than, 90%, 95% or 99% to these in a method according to claim 1.

Description

FIELD OF THE INVENTION The present invention is in the field of molecular biology, diagnostics, more particularly in the field of analytical and forensic sciences. The invention is further in the field of nucleic acid amplification and quantification, more particularly in the field of DNA quantification. BACKGROUND OF THE INVENTION The determination of the quantity of DNA recovered from forensic samples as well as other samples is a critical step in the overall DNA typing process, but also in the detection of DNA in various other fields of science. A narrow range of input DNA from 0.5 to 2 ng is often needed to produce optimal results with for example multiplex DNA typing kits. Therefore, in order to ensure that a positive result is a positive result and/or a negative result is a negative result due to the absence of DNA, quantification of DNA is of absolute importance. Furthermore, the quality of standards for forensic DNA testing laboratories requires human-specific DNA quantification. This is due to isolation techniques that can recover human DNA as well as bacterial and other exogenous DNA. A number of procedures have been developed to permit quantification of human-specific DNA including start-blot techniques, liquid based hybridization assays and real-time polymerase chain reaction (PCR). Currently, real-time PCR is the dominant technique due to its wide dynamic range and ease of automation. The modern short tandem repeat (STR) kits have become much more sensitive and can obtain good results even using low amounts of DNA. Therefore, there is a desire for a method, kit and nucleic acid region that allows precise and accurate quantification of human DNA even in low concentrated samples. There are certain quantification and detection kits already available. One such kit is the Quantifiler Human Kit (Applied Biosystems) another kit is Quantifiler Duo Kit (Applied Biosystems) another kit is the Plexor HY Real-Time PCR Quantification Kit (Promega). Both the Quantifiler Duo Kit and the Plexor HY Kit target an autosomal and a gonosomal (Y-chromosome) target on the genome. However, the kits currently on the market present some drawbacks. According to LaSalle et al. (Forensic Science International: Genetics, (2011) 5: 185-193) the Quantifiler Kits are more accurate in the quantification but have a lower dynamic range as the Plexor HY. The Plexor HY offers a higher dynamic range due to the amplification of a multicopy target, but a lower accuracy. This lower accuracy can be attributed to the multicopy target. If less than the full set of 20 copies on a genome amplify, because of, for example, instability in the target copy number, than the ratio between the amplification between autosomal and gonosomal (Y) target may vary. The dynamic range of the Plexor HY kit is slightly better than that of the other kit (LaSalle et al., Forensic Science International: Genetics, (2011) 5: 185-193). In a statistical comparison, it has been demonstrated a significant difference between the two kits (LaSalle et al., Forensic Science International: Genetics, (2011) 5: 185-193). Another important parameter in forensics is the degradation grade of the DNA that has to be analyzed. Since the amplicon size of the Quantifiler Human and Plexor HY vary from 62 to 133 base pairs (bp), significant differences might be expected when the kits are applied to degraded DNA. Also, inhibitors must be taken into account. It may well be that DNA is present in the reaction no result is obtained due to the presence of inhibitory substances. In cases of sexual assault samples, a quantification of the DNA is challenging, due to the presence of DNA molecules from both, the female victim as well as the male attacker. Furthermore, in a typical sample, the amount of female DNA exceeds the amount of male DNA by several orders of magnitude. Thus, a sensitive, male specific DNA quantification method which can accurately detect and quantify male DNA even in a high background of female DNA, is therefore of great interest. Reported herein is a qPCR-based DNA quantification system that is highly sensitive to detect low amounts of male DNA in a high background of female DNA and assess in parallel the male DNA degradation and/or integrity of the male DNA. Document WO2012113577 discloses amplification-based method of analysing DNA in a sample, by targeting MCL-Y locus. SUMMARY OF THE INVENTION The invention relates to a method for detecting and/or quantifying DNA, in particular the fraction of male DNA in a sample, (i) wherein the method comprises the step of amplification of a multicopy locus within the Y-chromosome (MCL-Y), wherein said locus shares at least 85% sequence identity to a sequence according to SEQ ID NO. 3 over a stretch of at least 60 base pairs (bp) or with the reverse complement thereof or, (ii) wherein the locus is amplifiable with a primer pair according to SEQ ID NO. 1 and SEQ ID NO. 2 or the reverse complement thereof, and further characterized