Methods for simultaneous amplification of target loci

• US 9,677,118 B2
• Filed: 11/24/2014
• Issued: 06/13/2017
• Est. Priority Date: 04/21/2014
• Status: Active Grant

• Current Assignee: Natera Incorporated (Natera)
• Original Assignee: Natera Incorporated (Natera)
• Inventors: Zimmermann, Bernhard, Lacroute, Philippe, Hill, Matthew, Wong, Alexander, Dodd, Michael
• Primary Examiner(s): MUMMERT, STEPHANIE KANE
• Application Number:
  US14/538,982
• Publication Number:
  US 20150322507A1
• Time in Patent Office: 932 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 human loci with a library of at least 50 non-immobilized, non-identical primers that simultaneously hybridize to at least 50 non-identical target human loci to produce a reaction mixture;

wherein the primers do not include molecular inversion probes (MIPs);

(b) 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 non-identical primers;

wherein the length of the annealing step of the reaction conditions is greater than 5 minutes; and

wherein at least 50 non-identical target human loci are simultaneously amplified; and

(c) sequencing the amplified products.

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

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

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

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

4. The method of claim 3, wherein the annealing temperature is at least 8°

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

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

C. greater than the average melting temperature of the at least 50 non-identical primers.

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

C. greater than the average melting temperature of the at least 50 non-identical primers.

7. The method of claim 1, wherein the range of melting temperature of the at least 50 non-identical primers is between 1 to 5°

C., inclusive.

8. The method of claim 1, 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 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, further comprising performing universal amplification on nucleic acids in the sample prior to step (a).

11. The method of claim 1, wherein at least 50 of the primers have at least 90% identity to the corresponding region of SEQ ID Nos. 1-44,610.

12. The method of claim 1, wherein the library comprises at least 500 non-immobilized, non-identical primers that simultaneously hybridize to the at least 500 non-identical target human loci, wherein the annealing temperature for the reaction conditions is greater than a melting temperature of the at least 500 non-identical primers, and wherein at least 500 non-identical target human loci are simultaneously amplified.

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

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

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

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

17. The method of claim 1, wherein the at least 50 non-identical primers have 2, 1, or 0 guanines or cytosines in the last 5 bases at the 3′

end of the primers.

18. The method of claim 1, wherein the at least 50 non-identical primers comprise a 5′

region that is not specific for a target human locus followed by a region that is specific for a target human locus, an internal region that is not specific for the target human locus and forms a loop structure, and a 3′

region that is specific for the target human locus.

19. The method of claim 1, wherein the sample comprises maternal DNA from the pregnant mother of a fetus and fetal DNA, and wherein the method further comprises determining the presence or absence of a fetal chromosome abnormality from the sequencing data.

20. The method of claim 1, wherein the sample comprises DNA from a single cell.

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

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

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

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

23. 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 reaction conditions is greater than a melting temperature of the at least 1000 non-identical primers, and wherein the at least 1000 non-identical target human loci are simultaneously amplified.

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