Methods for simultaneous amplification of target loci

• US 10,597,709 B2
• Filed: 05/14/2019
• Issued: 03/24/2020
• Est. Priority Date: 04/21/2014
• Status: Active Grant

• Current Assignee: Natera Incorporated (Natera)
• Original Assignee: Natera Incorporated (Natera)
• Inventors: Zimmermann, Bernhard, Hill, Matthew, Lacroute, Philippe, Dodd, Michael, Wong, Alexander
• Primary Examiner(s): Mummert, Stephanie K
• Application Number:
  US16/412,301
• Publication Number:
  US 20190316177A1
• Time in Patent Office: 315 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• 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 1/686 Polymerase chain reaction [PCR]
• 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 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 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 of amplifying target loci in a nucleic acid sample, the method comprising:

contacting the nucleic acid sample comprising at least 50 target loci with a library of at least 50 different primers to produce a reaction mixture;

subjecting the reaction mixture to primer extension reaction conditions to produce amplified products comprising target amplicons;

wherein the annealing temperature for the reaction conditions is greater than a melting temperature of the at least 50 primers, and wherein the at least 50 of the target loci are simultaneously amplified; and

sequencing the amplified products.

2. The method of claim 1, wherein the target loci are SNP loci.

3. The method of claim 1, wherein the annealing temperature is at least 3°

C. greater than the melting temperature of the primers.

4. The method of claim 1, wherein the annealing temperature is at least 3°

C. greater than the highest melting temperature of the primers.

5. The method of claim 1, wherein the annealing temperature is at least 8°

C. greater than the highest melting temperature of the primers.

6. The method of claim 1, wherein the annealing temperature is at least 3°

C. greater than the average melting temperature of the primers.

7. The method of claim 1, wherein the annealing temperature is at least 8°

C. greater than the average melting temperature of the primers.

8. The method of claim 1, wherein the range of melting temperatures of the primers is less than 5°

C.

9. The method of claim 1, wherein the Δ

G values for each possible combination of two primers in the library are all equal to or greater than −

5 kcal/mol.

10. The method of claim 1, wherein at least 100 target loci are amplified in a single reaction mixture.

11. The method of claim 1, wherein at least 200 target loci are amplified in a single reaction mixture.

12. The method of claim 1, wherein at least 90% of the amplified products are target amplicons.

13. The method of claim 1, wherein at least 90% of the target loci are amplified.

14. The method of claim 1, wherein less than 20% of the amplified products are primer dimers.

15. The method of claim 1, wherein the concentration of each primer in the library is less than 20 nM.

16. The method of claim 1, wherein the primers have 2, 1, or 0 guanines or cytosines in the last 5 bases at the 3′

end of the primers.

17. The method of claim 1, wherein the length of the annealing step of the reaction conditions is 3-60 minutes.

18. The method of claim 1, wherein the target loci are SNP loci, and wherein the nucleic acid sample comprises cell-free DNA of mixed origin.

19. The method of claim 18, wherein the nucleic acid sample comprises cell-free DNA from a transplant.

20. The method of claim 18, wherein the nucleic acid sample is from a transplant recipient.