Method for quantifying human DNA

Patent metadata

Field	Value
Publication number	US-9290815-B2
Application number	US-201214000096-A
Country	US
Kind code	B2
Filing date	Feb 21, 2012
Priority date	Feb 21, 2011
Publication date	Mar 22, 2016
Grant date	Mar 22, 2016

Abstract

The invention provides for a method for quantifying one or more nucleic acids of a genome in a sample comprising the steps of, (a) amplifying a first nucleic acid to be quantified, (b) determining the amount of said first nucleic acid by comparison of the amount of amplification product from said first nucleic acid with at least one amplification product from a second template nucleic acid, (c) wherein said second template nucleic acid was generated using whole genome amplification and wherein the starting concentration of the second template nucleic acid is known.

First claim

The invention claimed is:

1. Method for quantifying one or more nucleic acids of a genome in a sample comprising the steps of,
a. amplifying a first nucleic acid to be quantified from a first template nucleic acid,
b. determining the amount of said first nucleic acid by comparison of the amount of a first amplification product from said first nucleic acid with at least one second amplification product from a second template nucleic acid,
c. wherein said second template nucleic acid was generated using whole genome amplification and wherein the starting concentration of the second template nucleic acid is known; and
wherein the second amplification product comprises a sequence at least 60% identical to SEQ ID NO: 1 over a stretch of 80 contiguous base pairs.

2. Method according to claim 1 , wherein the nucleic acid sequences of the first nucleic acid and second template nucleic acid are from the same organism.

3. Method according to claim 1 , wherein the nucleic acids are DNA.

4. Method according to claim 1 , wherein the nucleic acid sequences of the first and second template nucleic acid are of human origin.

5. Method according to claim 1 , wherein said second template nucleic acid is from a nucleic acid mixture from various individuals of a given type of organism.

6. Method according to claim 1 , wherein said second template nucleic acid was generated using two or more rounds of whole genome amplification.

7. Method according to claim 1 , wherein the comparison is done with two or more second amplification products from said second template nucleic acids, wherein the two or more second amplification products are generated from different starting concentrations of said second template nucleic acids.

8. Method according to claim 7 , wherein the different starting concentrations of said second template nucleic acid have been adjusted in such a way that they reflect a substantially linear range of concentrations.

9. Method according to claim 1 , wherein the second template nucleic acid comprises primarily human nucleic acid sequences and its origin is selected from one of the following samples: blood, whole blood, fractions of blood, isolated nucleated blood cells, buccal swabs, saliva, sputum, urine and biopsies.

10. Method according to claim 1 , wherein the amplification reaction of the first nucleic acid to be amplified comprises an internal control nucleic acid.

11. Method according to claim 1 , wherein the amplification process is a real-time amplification process.

12. Method according to claim 11 , wherein a Ct value is obtained from the real-time amplification process and is compared with a standard curve generated using different dilutions of the second template nucleic acid.

13. The method of claim 1 , wherein the second amplification product comprises a sequence at least 70% identical to SEQ ID NO: 1 over a stretch of 80 contiguous base pairs.

14. The method of claim 1 , wherein the second amplification product comprises a sequence at least 80% identical to SEQ ID NO: 1 over a stretch of 80 contiguous base pairs.

15. The method of claim 1 , wherein the second amplification product comprises a sequence at least 90% identical to SEQ ID NO: 1 over a stretch of 80 contiguous base pairs.

16. The method of claim 1 , wherein the second amplification product comprises a sequence at least 95% identical to SEQ ID NO: 1 over a stretch of 80 contiguous base pairs.

17. The method of claim 1 , wherein the second amplification product comprises a sequence at least 98% identical to SEQ ID NO: 1 over a stretch of 80 contiguous base pairs.

18. The method of claim 1 wherein the sequence is SEQ ID NO: 1.

19. The method of claim 1 , wherein the sequence at least 60% identical to SEQ ID NO: 1 over a stretch of 80 contiguous base pairs is present in at least 10 copies in the second template nucleic acid.

20. The method of claim 1 , wherein the sequence at least 60% identical to SEQ ID NO: 1 over a stretch of 80 contiguous base pairs is present in at least 20 copies in the second template nucleic acid.

21. The method of claim 1 , wherein the sequence at least 60% identical to SEQ ID NO: 1 over a stretch of 80 contiguous base pairs is present in at least 29 copies in the second template nucleic acid.

22. The method of claim 1 , wherein the sequence at least 60% identical to SEQ ID NO: 1 over a stretch of 80 contiguous base pairs is present on a human chromosome selected from the group consisting of chromosomes 1, 4, 5, 7, 11 and 16.

23. The method of claim 1 , wherein a primer is used to amplify the sequence at least 60% identical to SEQ ID NO: 1 over a stretch of 80 contiguous base pairs and wherein the primer is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.

24. The method of claim 1 , wherein a primer is used to amplify the sequence at least 60% identical to SEQ ID NO: 1 over a stretch of 80 contiguous base pairs and wherein the primer differs from a polynucleotide selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12; by no more than 5 nucleotides over a stretch of 18 contiguous nucleotides.

25. The method of claim 1 , wherein two primers are used to amplify the sequence at least 60% identical to SEQ ID NO: 1 over a stretch of 80 contiguous base pairs and wherein the primers are SEQ ID NO: 11 and SEQ ID NO: 12.

Assignees

• Di Pasquale Francesca
• Engel Holger
• Qiagen Gmbh

Inventors

• Di Pasquale Francesca
• Engel Holger

Classifications

• C12Q1/6851 Primary

  Quantitative amplification · CPC title

• C12Q2600/166

  Oligonucleotides used as internal standards, controls or normalisation probes · CPC title

• C12Q1/6876

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• C12Q1/6888 Primary

  for detection or identification of organisms · CPC title

• C12Q2545/113

  with an external standard/control, i.e. control reaction is separated from the test/target reaction · CPC title