Systems and methods for allele enrichment using multiplexed blocker displacement amplification

Patent metadata

Field	Value
Publication number	US-12331350-B2
Application number	US-201916971411-A
Country	US
Kind code	B2
Filing date	Feb 20, 2019
Priority date	Feb 20, 2018
Publication date	Jun 17, 2025
Grant date	Jun 17, 2025

Abstract

Provided herein are reagents and methods for simultaneously enriching many potential rare genetic variants at different genetic loci. The rare variants enriched can include single nucleotide polymorphisms (SNPs), single nucleotide variants, or small insertions and deletions. Embodiments of the invention include procedures for integration with downstream next generation sequencing (NGS) analysis. Embodiments of the invention include analysis of nonpathogenic SNPs for the determination of cell identity and detection of cell contamination using qPCR or NGS.

First claim

What is claimed is:

1. A method for simultaneously amplifying and detecting allelic variants at at least ten genetic loci, the method comprising:
(a) mixing a sample comprising DNA with a DNA polymerase and a blocker displacement amplification (BDA) oligo set for each genetic locus, each BDA oligo set comprising (i) a BDA forward primer, (ii) a BDA blocker, and (iii) a BDA reverse primer, wherein at least four nucleotides at the 3′ end of each BDA forward primer sequence are also present at or near the 5′ end of its respective BDA blocker sequence, wherein each BDA blocker contains a 3′ sequence or modification that prevents extension by DNA polymerase, and wherein the concentration of each BDA blocker is at least twice that of its respective BDA forward primer; and
(b) subjecting the mixture to at least four cycles of amplification, thereby producing amplicons;
(c) performing next-generation sequencing (NGS) of the amplicons.

2. The method of claim 1 , wherein the DNA polymerase has 3′ to 5′ exonuclease activity.

3. The method of claim 2 , wherein each BDA blocker has a 3′ modification that prevents 3′ to 5′ exonuclease activity.

4. The method of claim 1 , wherein the concentration of each BDA reverse primer and/or each BDA forward primer is determined based on a reads analysis of a previous calibration NGS experiment, wherein the concentration of each BDA reverse primer and/or each BDA forward primer is increased relative to the concentration used for the previous calibration NGS experiment.

5. The method of claim 4 , wherein the concentration of each BDA reverse primer follows a formula: [rP]new=[rP]old * (Reads_median/Reads_amplicon){circumflex over ( )}X, where [rP]old is the previous concentration of the reverse primer, Reads_median is the median reads mapped to each amplicon, Reads_amplicon is the reads mapped to the amplicon corresponding to said reverse primer, and X is an adjustment factor between 0.25 and 1.

6. The method of claim 4 , wherein the concentration of each BDA forward primer follows a formula: [fP]new=[fP]old * (Reads_median/Reads_amplicon){circumflex over ( )}X, where [fP]old is the previous concentration of the forward primer, Reads_median is the median reads mapped to each amplicon, Reads_amplicon is the reads mapped to the amplicon corresponding to said forward primer, and X is an adjustment factor between 0.25 and 1.

7. The method of claim 1 , wherein the BDA oligo set comprises at least 10 BDA oligo sets, each BDA oligo set comprising (i) a BDA forward primer, (ii) a BDA blocker, and (iii) a BDA reverse primer, wherein at least four nucleotides at the 3′ end of each BDA forward primer sequence are also present at or near the 5′ end of its corresponding BDA blocker sequence, wherein each BDA blocker contains a 3′ sequence or modification that prevents extension by DNA polymerase, and wherein the concentration of each BDA blocker is at least twice that of its corresponding BDA forward primer, wherein each BDA blocker is complementary to a genomic region bearing a single nucleotide polymorphism (SNP) in which the alternative allele has a population frequency of between 10% and 90%, and wherein each corresponding BDA forward primer is not complementary to the SNP locus.

8. The method of claim 7 , wherein none of the BDA forward primers and none of the BDA reverse primers are complementary to any SNP in which the alternative allele has a population frequency of over 1%.

9. The method of claim 7 , wherein the genomic position that each BDA reverse primer binds is located between 100 nt and 500 nt away from the genomic position that its corresponding BDA forward primer binds.

10. The method of claim 7 , wherein the calculated ΔG°'s for each BDA forward primer binding to its corresponding complement are all within 2 kcal/mol of each other at 60° C. in 0.18 M Na + .

11. The method of claim 7 , wherein the calculated ΔG° for each BDA blocker binding to its corresponding complement is between 0.5 kcal/mol and 3.5kcal/mol more favorable than the ΔG° of binding between the corresponding BDA forward primer and its complement at 60° C. in 0.18 M Na + .

12. A method for detecting contamination of a base cell line, the method comprising:
(a) extracting genomic DNA from a cell sample;
(b) producing and detecting amplicons according to the method of claim 1
(c) further detecting contamination, wherein the further detecting comprises identifying the presence of amplicons that are not amplicons from the base cell line.

Assignees

• Univ Rice William M

Inventors

• Zhang David Yu
• Song Ping
• Chen Xi

Classifications

• C12Q1/6858 Primary

  Allele-specific amplification · CPC title