Methods for simultaneous amplification of target loci

• US 10,655,180 B2
• Filed: 03/14/2019
• Issued: 05/19/2020
• Est. Priority Date: 05/18/2010
• Status: Active Grant

• Current Assignee: Natera Incorporated (Natera)
• Original Assignee: Natera Incorporated (Natera)
• Inventors: Babiarz, Joshua, Constantin, Tudor Pompiliu, Eubank, Lane A., Gemelos, George, Hill, Matthew Micah, Kirkizlar, Huseyin Eser, Rabinowitz, Matthew, Sakarya, Onur, Sigurjonsson, Styrmir, Zimmermann, Bernhard
• Primary Examiner(s): Mummert, Stephanie K
• Application Number:
  US16/353,636
• Publication Number:
  US 20190203294A1
• Time in Patent Office: 432 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2537/159 Reduction of complexity, e.g. amplification of subsets, removing duplicated genomic regions
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

The invention provides methods for simultaneously amplifying multiple nucleic acid regions of interest in one reaction volume as well as methods for selecting a library of primers for use in such amplification methods. The invention also provides library of primers with desirable characteristics, such as minimal formation of amplified primer dimers or other non-target amplicons.

Claims

1. A method for measuring an amount of DNA in a biological sample, comprising:

(a) extracting cell-free DNA of mixed origin from a biological sample of a subject, wherein the cell-free DNA comprises DNA from the subject and DNA from a genetically distinct individual, wherein neither the subject nor the genetically distinct individual is a fetus, and wherein the DNA of mixed origin comprises DNA from a transplant;

(b) performing targeted PCR amplification of the cell-free DNA at more than 100 SNP loci in a single reaction volume using more than 100 PCR primer pairs, wherein the amplified SNP loci comprise SNP loci on at least chromosome 1, 2, or 3;

(c) determining the genotypes of the amplified SNP loci and measuring an amount of one or more alleles at the SNP loci, wherein the genotypes of the amplified SNP loci are determined by high-throughput sequencing; and

(d) measuring an amount of the DNA from the genetically distinct individual present in the biological sample using the amount of one or more alleles at the SNP loci,

wherein the method is performed without prior knowledge of genotypes of the genetically distinct individual.

2. The method of claim 1, wherein the biological sample is a blood, serum, plasma, or urine sample.

3. The method of claim 1, wherein the extracting step comprises size selection to enrich for shorter cell-free DNA.

4. The method of claim 1, wherein the primer pairs are each designed to amplify less than about 100 bp of DNA.

5. The method of claim 1, wherein the primer pairs are each designed to amplify less than about 80 bp of DNA.

6. The method of claim 1, wherein the primer pairs are each designed to amplify about 65-80 bp of DNA.

7. The method of claim 1, wherein more than 200 SNP loci are amplified in a single reaction volume.

8. The method of claim 1, wherein more than 500 SNP loci are amplified in a single reaction volume.

9. The method of claim 1, wherein more than 1000 SNP loci are amplified in a single reaction volume.

10. The method of claim 1, wherein more than 2000 SNP loci are amplified in a single reaction volume.

11. The method of claim 1, wherein the amplified SNP loci comprise SNP loci on chromosome 1.

12. The method of claim 1, wherein the amplified SNP loci comprise SNP loci on chromosome 2.

13. The method of claim 1, wherein the amplified SNP loci comprise SNP loci on chromosome 3.

14. A method for measuring an amount of DNA in a biological sample, the method comprising:

(a) performing a targeted PCR amplification for more than 100 SNP loci on one or more chromosomes expected to be disomic in a single reaction mixture using more than 100 PCR primer pairs, wherein the reaction mixture comprises cell-free DNA extracted from a biological sample of a subject comprising DNA of mixed origin, wherein the DNA of mixed origin comprises DNA from the subject and DNA from a genetically distinct individual, wherein neither the subject nor the genetically distinct individual is a fetus, wherein the DNA of mixed origin comprises DNA from a transplant, and wherein the amplified SNP loci comprise SNP loci on at least chromosome 1, 2, or 3;

(b) measuring a quantity of each allele at a plurality of amplified SNP loci that comprise an allele present in the genetically distinct individual but not the subject, wherein the quantity of each allele at a plurality of amplified SNP loci are measured by high-throughput sequencing;

(c) measuring an amount of the DNA from the genetically distinct individual in the biological sample using the quantity of each allele at the SNP loci and an expected quantity of each allele at the SNP loci for different DNA fractions,

wherein the method is performed without prior knowledge of genotypes of the genetically distinct individual.

15. The method of claim 14, further comprising determining a bias of the PCR amplification, and using the bias to statistically correct the determined quantity of each allele at the plurality of SNP loci on the one or more chromosomes expected to be disomic before the quantity of each allele is used to determine the amount of the DNA from the genetically distinct individual.

16. The method of claim 14, wherein the biological sample is a blood, serum, plasma, or urine sample, wherein more than 500 SNP loci are amplified in a single reaction volume.