	US 11,718,847 B2
	Amplifying oligonucleotides and producing libraries of dual guide constructs
Jeffrey Carl Braman, Carlsbad, CA (US); Peter James Sheffield, Vista, CA (US); and Holly Hogrefe, San Diego, CA (US)
Assigned to Agilent Technologies, Inc., Santa Clara, CA (US)
Filed by Agilent Technologies, Inc., Santa Clara, CA (US)
Filed on Aug. 29, 2018, as Appl. No. 16/116,147.
Prior Publication US 2020/0071690 A1, Mar. 5, 2020
Int. Cl. C12N 15/10 (2006.01); C12N 15/11 (2006.01); C12Q 1/686 (2018.01); C12N 15/66 (2006.01); C12Q 1/6853 (2018.01); C40B 50/06 (2006.01); C40B 50/04 (2006.01); C40B 70/00 (2006.01)

CPC C12N 15/1065 (2013.01) [C12N 15/1068 (2013.01); C12N 15/1096 (2013.01); C12N 15/11 (2013.01); C12N 15/66 (2013.01); C12Q 1/686 (2013.01); C12Q 1/6853 (2013.01); C12N 2330/31 (2013.01); C40B 50/04 (2013.01); C40B 50/06 (2013.01); C40B 70/00 (2013.01)]

17 Claims

1. A method of amplifying a pool of oligonucleotides comprising:

providing a pool of oligonucleotides, wherein each of the oligonucleotides comprises a defined pair of sequences encoding guide RNA segments;

forming an amplification mixture comprising the pool of oligonucleotides, an amplification enzyme, deoxyribonucleotide triphosphates (dNTPs), and primers;

thermocycling the amplification mixture between a denaturing temperature, an annealing temperature, and an extension temperature, a sufficient number of times to produce a library of oligonucleotide library members, wherein the amplification mixture comprises DMSO in an amount from 3% to 6.5% v/v;

wherein at least 99.9% of the library members have one of the defined pairs of sequences encoding guide RNA segments.