Methods for simultaneous amplification of target loci

• US 10,351,906 B2
• Filed: 10/27/2016
• Issued: 07/16/2019
• 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:
  US15/336,630
• Publication Number:
  US 20170145474A1
• Time in Patent Office: 992 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:

(a) contacting a sample comprising target loci with a library of at least 50 non-immobilized, non-identical PCR primers that simultaneously hybridize to at least 50 non-identical target loci to produce a reaction mixture; and

(b) subjecting the reaction mixture to PCR conditions to produce amplified products comprising target amplicons;

wherein an annealing temperature for the PCR conditions is greater than a melting temperature for at least 95% of the at least 50 non-identical PCR primers;

wherein a length of an annealing step of the PCR conditions is greater than 5 minutes; and

wherein at least 50 non-identical target loci are simultaneously amplified.

2. The method of claim 1, wherein the lengths of the target amplicons are between 30 and 200 nucleotides and wherein the concentration of each primer in the library is less than 20 nM.

3. The method of claim 1, wherein the annealing temperature is 1 to 15°

C. greater than the highest melting temperature of at least 95% of the at least 50 non-identical primers.

4. The method of claim 1, wherein the library comprises at least 1000 non-immobilized, non-identical primer pairs that simultaneously hybridize to at least 1000 non-identical target human loci, wherein the annealing temperature for the PCR conditions is greater than a melting temperature of at least 90% of the at least 1000 non-identical primers, and wherein the at least 1000 non-identical target loci are simultaneously amplified.

5. The method of claim 1, wherein the length of the annealing step of the PCR conditions is between 10 and 60 minutes.

6. The method of claim 1, wherein the AG values for each possible combination of two primers in the library are all equal to or greater than −

5 kcal/mol.

7. The method of claim 1, further comprising performing universal amplification on nucleic acids in the sample prior to step (a), and further comprising sequencing the amplified products comprising target amplicons after step (b).

8. The method of claim 1, wherein at least 90% of the amplified products are target amplicons, and wherein the range of melting temperatures of the at least 50 non-identical primers is less than 5°

C.

9. The method of claim 1, wherein at least 90% of the target loci are amplified and wherein less than 20% of the amplified products are primer dimers.

10. A method of amplifying target loci in a nucleic acid sample, the method comprising:

(a) contacting a sample comprising target loci with a library of at least 50 non-immobilized, non-identical PCR primers that simultaneously hybridize to at least 50 non-identical target loci to produce a reaction mixture, wherein the sample is derived from blood; and

(b) subjecting the reaction mixture to PCR conditions to produce amplified products comprising target amplicons, wherein the annealing temperature for the PCR conditions is greater than a melting temperature for at least 95% of the at least 50 non-identical PCR primers, wherein the melting temperature is a calculated temperature at which one-half of a DNA duplex of a primer and its perfect complement is predicted to dissociate and become single stranded DNA, wherein the length of an annealing step of the PCR conditions is between 5 and 60 minutes, wherein the length of the target amplicons is between 30 and 200 nucleotides, and wherein at least 50 non-identical target human loci are simultaneously amplified.

11. The method of claim 10, wherein the sample is from an individual suspected of having cancer.

12. The method of claim 10, wherein the sample comprises maternal DNA from the pregnant mother of a fetus and fetal DNA.

13. The method of claim 10, wherein the melting temperature of at least 90% of the primers is between 54 and 60.5, inclusive, and the annealing temperature is between 60°

C. and 70°

C., inclusive.

14. The method of claim 10, wherein the annealing temperature is 1 to 10°

C. greater than the highest melting temperature of at least 95% of the at least 50 non-identical primers.

15. The method of claim 10, wherein the library comprises between 10,000 and 100,000 non-immobilized, non-identical primers or primer pairs that simultaneously hybridize to the at least 10,000 and 100,000 non-identical target human loci, wherein the annealing temperature for the PCR conditions is greater than a melting temperature of at least 90% of the between 10,000 and 100,000 non-identical primers, and wherein between 10,000 and 100,000 non-identical target human loci are simultaneously amplified.

16. A method of amplifying target loci in a nucleic acid sample, the method comprising:

(a) contacting a sample comprising target loci with a library of between 10,000 and 100,000 non-immobilized, non-identical PCR primers or primer pairs that simultaneously hybridize to between 10,000 and 100,000 non-identical target loci to produce a reaction mixture, wherein the concentration of each primer in the reaction mixture is less than 20 nM; and

(b) subjecting the reaction mixture to PCR conditions to produce amplified products comprising target amplicons, wherein an annealing temperature for the PCR conditions is greater than a melting temperature for at least 95% of primers of the 10,000 to 100,000 non-identical PCR primers or primer pairs, wherein the length of an annealing step of the PCR conditions is between 5 and 60 minutes, and wherein between 10,000 and 100,000 non-identical target loci are simultaneously amplified.

17. The method of claim 16, wherein the melting temperature of at least 90% of primers of the PCR primers or primer pairs is between 50°

C. and 60.5°

C., inclusive, and the annealing temperature is between 60°

C. and 70°

C., inclusive.

18. The method of claim 16, wherein the length of at least 95% of primers of the PCR primers or primer pairs is between 15 to 40 nucleotides, inclusive, the length of at least 95% of the target amplicons is between 30 and 200 nucleotides, and the concentration of at least 95% of primers of the PCR primers or primer pairs is less than 10 nm.

19. The method of claim 16, wherein the annealing temperature is 1 to 10°

C. greater than the highest melting temperature of at least 95% of primers of the PCR primers or primer pairs.

20. The method of claim 16, wherein the annealing temperature is at least 3°

C. greater than the highest melting temperature of at least 90% of primers of the PCR primers or primer pairs.