Granted Patent B2

US 12,655,483 · App. 18/243,593 · Granted Jun 16, 2026

Inventors: Matthew Rabinowitz (San Francisco, CA); Matthew Hill (Belmont, CA); Bernhard Zimmermann (Manteca, CA); George Gemelos (Portland, OR); Johan Baner (San Francisco, CA); Milena Banjevic (Los Altos Hills, CA); Allison Ryan (Belmont, CA); Styrmir Sigurjonsson (San Jose, CA); Zachary Demko (San Francisco, CA)

Assignee: Natera, Inc.

C12Q1/6883C12Q1/6809C12Q1/6811C12Q1/6844C12Q1/6848C12Q1/6851C12Q1/6855C12Q1/6869C12Q1/6874C12Q2600/156

Abstract

Claims (22)

1 . A method for preparing non-naturally occurring compositions of amplified DNA from a biological sample obtained from a second individual, wherein the sample contains DNA from a first individual, the method comprising:

(a) extracting cell-free DNA of mixed origin from the biological sample, wherein said extracted cell-free DNA of mixed origin from said biological sample includes DNA from the first individual and DNA from the second individual, and wherein the biological sample is a blood, serum, plasma, or urine sample;

(b) performing a multiplex targeted amplification, thereby amplifying at least 50 loci of the said extracted cell-free DNA from said biological sample in a single reaction volume and generating a first population of amplicons; and

(c) performing a second amplification to obtain a second population of amplicons, wherein at least some of the amplicons of the second population contain a barcode and a sequencing adapter.

2 . The method of claim 1 , wherein at least some of the amplicons in said second population of amplicons are sequenced to generate genetic data.

3 . The method of claim 2 , wherein at least some of said genetic data is used to determine an amount of an allele at each of 50 or more of the loci.

4 . The method of claim 3 , wherein the method further comprises determining an amount of DNA from said first individual in the biological sample obtained from said second individual.

5 . The method of claim 4 , wherein the first individual is a fetus and the second individual is a pregnant woman, wherein determining an amount of DNA from said first individual includes determining the fetal fraction of the cell-free DNA in the biological sample using the amount of each allele at some or all of the loci.

6 . The method of claim 4 , wherein determining an amount of DNA from said first individual includes determining a relative amount or an absolute amount of DNA.

7 . The method of claim 1 , wherein the multiplex targeted amplification includes amplifying target sequences not exceeding 100 bp in length using a plurality of primer pairs, and thereby generating a plurality of amplicons, wherein at least some of the target sequences include the said at least 50 loci.

8 . The method of claim 1 , wherein the multiplex targeted amplification includes amplifying target sequences not exceeding 80 bp in length using a plurality of primer pairs, and thereby generating a plurality of amplicons, wherein at least some of the target sequences include the said at least 50 loci.

9 . The method of claim 1 , wherein the multiplex targeted amplification includes amplifying target sequences 65-80 bp in length using a plurality of primer pairs, and thereby generating a plurality of amplicons, wherein at least some of the target sequences include the said at least 50 loci.

10 . The method of claim 1 , wherein the multiplex targeted amplification comprises 10-30 PCR cycles.

11 . The method of claim 1 , wherein the multiplex targeted amplification comprises amplifying 50-5,000 loci in a single reaction volume.

12 . The method of claim 1 , wherein the multiplex targeted amplification comprises amplifying 50-500 loci in a single reaction volume.

13 . The method of claim 1 , wherein the multiplex targeted amplification comprises amplifying 50-100 loci in a single reaction volume.

14 . The method of claim 1 , wherein the loci comprise single nucleotide polymorphism or variant loci.

15 . The method of claim 2 , wherein the sequencing is high-throughput sequencing.

16 . The method of claim 2 , wherein the sequencing is sequencing-by-synthesis.

17 . The method of claim 1 , wherein the multiplex targeted amplification is a PCR reaction performed with at least 50 different forward and reverse primer pairs.

18 . The method of claim 17 , wherein the PCR reaction comprises an annealing step having an annealing time that is greater than 3 minutes and an annealing temperature that is greater than a melting temperature of a plurality of the primers.

19 . The method of claim 1 , wherein the method does not comprise PCR amplification in a microfluidic device.

Assignments (2)

Continuity (34)

References Cited (400)