Methods for simultaneous amplification of target loci

• US 10,316,362 B2
• Filed: 10/20/2015
• Issued: 06/11/2019
• 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:
  US14/918,544
• Publication Number:
  US 20160369333A1
• Time in Patent Office: 1,330 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 of amplifying target loci of DNA molecules in a nucleic acid sample, the method comprising:

(a) contacting the sample and a library of at least 1,000 non-immobilized, non-identical primers that hybridize to at least 1,000 non-identical target loci to produce a reaction mixture in which the concentration of each primer is less than 20 nM;

wherein the average length of the DNA molecules is less than 200 base pairs; and

wherein a range of melting temperatures of the at least 1000 non-identical primers is less than 10°

C.;

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

wherein a length of an annealing step of the PCR conditions is greater than 10 minutes;

wherein the length of the target amplicons is less than 100 nucleotides;

wherein at least 1,000 different target human loci are amplified, and wherein at least 50% of the amplified products comprise target loci.

2. The method of claim 1, wherein a range of melting temperatures of the at least 1000 non-identical primers is less than 5°

C.

3. The method of claim 1, further comprising performing universal amplification on DNA molecules in the sample prior to step (a).

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

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

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

7. The method of claim 1, wherein the at least 1,000 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 humanlocus.

8. The method of claim 1, wherein the sample comprises DNA from a pregnant mother and from her fetus, and wherein the method further comprises amplifying the amplified human loci and determining the presence or absence of a fetal chromosome abnormality from the sequencing data.

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

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

13. The method of claim 1, wherein the concentration of primers in the reaction volume are less than 10 nM.

14. The method of claim 1, wherein the concentration of primers in the reaction volume are less than 5 nM.

15. The method of claim 1, wherein the library comprises at least 7,500 non-immobilized, non-identical primers that hybridize to at least 7,500 non-identical target human loci, and wherein the 7,500 non-identical target human loci are amplified.

16. The method of claim 1, The library of claim 1, wherein a range of guanine-cytosine (GC) content among the different test primers in the library is less than 30%.

17. The method of claim 14, wherein a range of melting temperatures among the different primers in the library is less than 20°

C.

18. The method of claim 15, wherein a range of the length among the different target amplicons is less than 50 nucleotides.

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

(a) contacting the sample and a library of at least 1,000 non-immobilized, non-identical primers that hybridize to at least 1,000 non-identical target loci to produce a reaction mixture in which the concentration of each primer is less than 20 nM;

wherein the average length of the DNA molecules is less than 200 base pairs;

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

wherein a length of an annealing step of the PCR conditions is greater than 10 minutes;

wherein the length of the target amplicons is less than 100 nucleotides;

wherein at least 1,000 different target human loci are amplified, wherein at least 50% of the amplified products comprise target loci; and

wherein the sample comprises or is derived from maternal blood.

11. The method of claim 10, wherein the method further comprises sequencing the amplified human loci and making a genetic diagnosis, wherein the genetic diagnosis comprises detecting a gene mutation or detecting a ploidy state of the target human loci.

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

(a) contacting the sample and a library of at least 1,000 non-immobilized, non-identical primers that hybridize to at least 1,000 non-identical target loci to produce a reaction mixture in which the concentration of each primer is less than 20 nM;

wherein the average length of the DNA molecules is less than 200 base pairs;

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

wherein a length of an annealing step of the PCR conditions is greater than 10 minutes;

wherein the length of the target amplicons is less than 100 nucleotides;

wherein at least 1,000 different target human loci are amplified, wherein at least 50% of the amplified products comprise target loci; and

wherein the target human loci comprise single nucleotidepolymorphisms.