Methods for simultaneous amplification of target loci

• US 10,526,658 B2
• Filed: 05/14/2019
• Issued: 01/07/2020
• 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:
  US16/412,331
• Publication Number:
  US 20190271043A1
• Time in Patent Office: 238 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 from a first individual in a biological sample of a second individual, comprising:

pre-amplifying at least 50 polymorphic loci from cell-free DNA of mixed origin in a single reaction volume to obtain pre-amplified DNA, wherein the cell-free DNA is extracted from the biological sample and comprises DNA from the first individual and DNA from the second individual, wherein neither the first individual nor the second individual is a fetus;

dividing the pre-amplified DNA into multiple aliquots;

amplifying subpools of the polymorphic loci in parallel in individual reaction volumes to obtain amplified DNA, wherein each reaction volume comprises at least one aliquot of the pre-amplified DNA; and

pooling the amplified DNA into one pool;

sequencing the amplified DNA by high-throughput sequencing and measuring an amount of each allele at the polymorphic loci; and

determining the amount of the DNA from the first individual in the biological sample using the amount of each allele at the polymorphic loci.

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 cell-free DNA of mixed origin comprises DNA from a transplant.

4. The method of claim 1, wherein the pre-amplifying step comprises 10-30 PCR cycles.

5. The method of claim 1, wherein the pre-amplifying step comprises 15 PCR cycles.

6. The method of claim 1, wherein 50-5,000 polymorphic loci are pre-amplified in a single reaction volume.

7. The method of claim 1, wherein 50-500 polymorphic loci are pre-amplified in a single reaction volume.

8. The method of claim 1, wherein more than 100 polymorphic loci are pre-amplified in a single reaction volume.

9. The method of claim 1, wherein more than 200 polymorphic loci are pre-amplified in a single reaction volume.

10. The method of claim 1, wherein the polymorphic loci are SNP loci.

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

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

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

14. The method of claim 1, wherein the sequencing is sequencing-by-synthesis.

15. The method of claim 1, wherein the method is performed without prior knowledge of genotypes of the first and second individuals.