# US 11,078,527 B2 · Method for quantifying and/or detecting human male DNA

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- Number: US 11,078,527 B2
- Document ID: US11078527B2
- Status: Granted
- Kind: B2
- Application number: 16/333,076
- Filed: 2017-09-15
- Published: 2019-08-15
- Granted: 2021-08-03
- Earliest claimed: 2016-09-22
- Assignees: Qiagen GmbH
- Applicants: QIAGEN GmbH
- Inventors: Miroslav Vranes; Ralf Peist; Mario Scherer; Stefan Otto Cornelius; Margaretha König
- Web page: https://patentgrep.com/patent/US11078527B2

## CPC

- C12Q 1/6827: for detection of mutation or polymorphism
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6851: Quantitative amplification
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q 1/6879: for sex determination
- C12Q 2525/151: repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
- C12Q 2600/16: Primer sets for multiplex assays
- C12Q 2600/166: Oligonucleotides used as internal standards, controls or normalisation probes
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

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.

## Claims

### Claim 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.

### Claim 2 (depends on 1)

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.

### Claim 3 (depends on 1)

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.

### Claim 4 (depends on 1)

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.

### Claim 5 (depends on 1)

The method according to claim 1, wherein said sample comprises male and female genomic DNA.

### Claim 6 (depends on 1)

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.

### Claim 7 (depends on 6)

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.

### Claim 8 (depends on 6)

The method according to claim 6, wherein the amplification reaction comprises amplifying at least two overlapping regions using at least one common primer.