Method for quantifying and/or detecting human male DNA
According to a first aspect of the present invention, a method is provided for detecting and/or quantifying male genomic DNA in a sample, wherein the method comprises the step of amplification of a multicopy locus within the human Y-chromosome (MCL-Y), wherein said locus shares at least 80% sequence identity to a sequence according to SEQ ID NO. 3 over a stretch of at least 60 base pairs (bp). A second aspect of the present invention relates to a primer or primer pair which hybridizes under stringent conditions to a sequence according to SEQ ID NO. 3 and/or any of 4 to 11. The invention also relates to a kit.
1. A method for detecting, assessing the status of and/or quantifying the fraction of male DNA in a sample, wherein the method comprises amplification of a multicopy locus within the Y-chromosome (MCL-Y), and wherein the amplification is performed using at least one primer selected from the group consisting of:
a. SEQ ID NO. 1;
b. SEQ ID NO. 2;
c. the reverse complement of SEQ ID NO. 1;
d. the reverse complement of SEQ ID NO. 2;
e. a primer that shares at least 90% sequence identity with SEQ ID NO. 1;
f. a primer that shares at least 90% sequence identity with SEQ ID NO. 2;
g. the reverse complement of a primer that shares at least 90% sequence identity with SEQ ID NO. 1; and
h. the reverse complement of a primer that shares at least 90% sequence identity with SEQ ID NO. 2.
2. The method according to claim 1 , wherein the amplification 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. the reverse complement of SEQ ID NO. 1 and the reverse complement of SEQ ID NO. 2;
c. 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; and
d. 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.
3. The method according to claim 1 , wherein the amplification step is performed using a primer pair having a sequence according to SEQ ID NO. 1 and SEQ ID NO. 2.
4. The method according to claim 1 , wherein said sample originates from whole blood, a blood fraction, an oral specimen, urine, human bioptic tissue or another part of a human body from which a genome is isolatable.
5. The method according to claim 1 , wherein said sample comprises male and female genomic DNA.
6. The method according to claim 1 , wherein the amplification step is performed by 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.
7. The method according to claim 6 , wherein the amplification reaction comprises any one or more of:
a. Tris-HCl at a pH of between 8 and 8.8;
b. a potassium salt selected from the group of potassium chloride and potassium sulfate;
c. an ammonium salt;
d. magnesium chloride; and
e. a hot-start polymerase.
8. The method according to claim 6 , wherein the amplification reaction comprises amplifying at least two overlapping regions using at least one common primer.