	US 12,331,350 B2
	Systems and methods for allele enrichment using multiplexed blocker displacement amplification
David Yu Zhang, Houston, TX (US); Ping Song, Houston, TX (US); and Xi Chen, Houston, TX (US)
Assigned to William Marsh Rice University, Houston, TX (US)
Appl. No. 16/971,411
Filed by William Marsh Rice University, Houston, TX (US)
PCT Filed Feb. 20, 2019, PCT No. PCT/US2019/018690 § 371(c)(1), (2) Date Aug. 20, 2020, PCT Pub. No. WO2019/164885, PCT Pub. Date Aug. 29, 2019.
Claims priority of provisional application 62/649,138, filed on Mar. 28, 2018.
Claims priority of provisional application 62/632,712, filed on Feb. 20, 2018.
Prior Publication US 2021/0024989 A1, Jan. 28, 2021
Int. Cl. C12Q 1/6858 (2018.01)

CPC C12Q 1/6858 (2013.01)

12 Claims

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;