# US 12,110,552 B2 · Methods for simultaneous amplification of target loci

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- Number: US 12,110,552 B2
- Document ID: US12110552B2
- Status: Granted
- Kind: B2
- Application number: 16/856,924
- Filed: 2020-04-23
- Published: 2020-10-08
- Granted: 2024-10-08
- Earliest claimed: 2010-05-18
- Assignees: Natera, Inc.
- Applicants: Natera, Inc.
- Inventors: Matthew Rabinowitz; Matthew Micah Hill; Bernhard Zimmermann; Johan Baner; George Gemelos; Milena Banjevic; Allison Ryan; Styrmir Sigurjonsson; Zachary Demko
- Web page: https://patentgrep.com/patent/US12110552B2

## CPC

- C12Q 1/6883: for diseases caused by alterations of genetic material
- C12Q 1/6809: Methods for determination or identification of nucleic acids involving differential detection
- C12Q 1/6811: Selection methods for production or design of target specific oligonucleotides or binding molecules
- C12Q 1/6844: Nucleic acid amplification reactions
- C12Q 1/6848: characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
- C12Q 1/6851: Quantitative amplification
- C12Q 1/6855: Ligating adaptors
- C12Q 1/6858: Allele-specific amplification
- C12Q 1/6869: Methods for sequencing
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12Q 2600/156: Polymorphic or mutational markers
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## 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

### Claim 1

A method for preparing a biological sample containing DNA from a first individual and a second individual, comprising:
isolating cell-free DNA from the biological sample, wherein the isolated cell-free DNA comprises the DNA from the first individual and DNA from the second individual;
amplifying the isolated cell-free DNA at a plurality of polymorphic loci on one or more chromosomes expected to be disomic, wherein the plurality of polymorphic loci comprises between 200 and 1,000 polymorphic loci, wherein the amplifying comprises amplifying the between 200 and 1,000 polymorphic loci from the cell-free DNA isolated from the biological sample using between 200 and 1,000 distinct primer pairs configure to target the between 200 and 1,000 polymorphic loci in a single reaction volume; and
preparing the isolated cell-free DNA amplified at the polymorphic loci for sequencing.

### Claim 2 (depends on 1)

The method of claim 1, wherein the method further comprises enriching the DNA in the sample at the plurality of polymorphic loci prior to the amplifying step.

### Claim 3 (depends on 2)

The method of claim 2, wherein the enrichment comprises:
obtaining a plurality of target-specific primers designed to hybridize to regions of DNA upstream and downstream of the polymorphic loci;
hybridizing the target-specific primers to the DNA; and
amplifying the DNA using the polymerase chain reaction to form amplicons.

### Claim 4 (depends on 2)

The method of claim 2, wherein the enrichment comprises preferentially enriching the DNA at the plurality of loci to minimize an average degree of allelic bias.

### Claim 5 (depends on 1)

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

### Claim 6 (depends on 5)

The method of claim 5, wherein the polymorphic loci comprise SNP loci on chromosome 1.

### Claim 7 (depends on 5)

The method of claim 5, wherein the polymorphic loci comprise SNP loci on chromosome 2.

### Claim 8 (depends on 5)

The method of claim 5, wherein the polymorphic loci comprise SNP loci on chromosome 3.

### Claim 9 (depends on 1)

The method of claim 1, wherein sequencing comprises high-throughput sequencing.

### Claim 10 (depends on 1)

The method of claim 1, wherein the cell-free DNA comprises DNA from a fetus or a transplant.

### Claim 11 (depends on 1)

The method of claim 1, wherein the biological sample is a blood sample.

### Claim 12 (depends on 1)

The method of claim 1, wherein the distinct primers are circularizing probes.

### Claim 13 (depends on 1)

The method of claim 1, wherein the distinct primers are configured to target the polymorphic loci via hybridization.

### Claim 14 (depends on 1)

The method of claim 1, wherein the distinct primers comprise PCR primers.

### Claim 15 (depends on 1)

The method of claim 1, wherein the total amount of cfDNA in the sample is between 10 pg and 100 pg.

### Claim 16 (depends on 15)

The method of claim 15, wherein less than or equal to 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, or 0.005% of the cfDNA molecules that have a first locus have a mutation in the first locus.

### Claim 17 (depends on 15)

The method of claim 15, wherein amplifying the isolated cell-free DNA comprises amplifying from 72%, 69%, 66%, 63%, 59%, or 56% of available cfDNA template fragment molecules.