US 12,435,360 B2
Reaction condition composition for circularizing oligonucleotide probes
Abraham Oommen, Lincoln, NE (US); Heather Piscatelli, Lincoln, NE (US); and Alyssa Hangman, Lincoln, NE (US)
Appl. No. 16/642,308
Filed by STEM ARTS PROJECTS, LLC, Lincoln, NE (US)
PCT Filed Jan. 11, 2019, PCT No. PCT/US2019/013222
§ 371(c)(1), (2) Date Feb. 26, 2020,
PCT Pub. No. WO2019/140211, PCT Pub. Date Jul. 18, 2019.
Claims priority of provisional application 62/616,866, filed on Jan. 12, 2018.
Prior Publication US 2020/0199661 A1, Jun. 25, 2020
Int. Cl. C12Q 1/6827 (2018.01)
CPC C12Q 1/6827 (2013.01) 17 Claims
 
1. A reaction condition composition for hybridization and ligation of circularizing oligonucleotide probes comprising:
a DNA ligase from 1 to 5 units;
a DNA polymerase from 0.2 to 2.5 units;
at least one circularizing oligonucleotide probe having a final concentration in the reaction condition composition after contact with a deoxyribose nucleic acid (DNA) sample from 0.0125 to 0.3 micromolar, wherein
the at least one circularizing oligonucleotide probe is specific to a first genomic variation;
a deoxyribonucleic acid buffer capable of maintaining a pH from 7 to 9;
NAD+ having a final concentration in the reaction condition composition after contact with the DNA sample from 0.1 to 1.4 millimolar;
at least two primers having a final concentration in the reaction condition composition after contact with the DNA sample from 0.1 to 0.5 micromolar, wherein the at least two primers are specific to a first replication sequence; and
deoxynucleotide triphosphates having a final contraction in the reaction condition composition after contact with the DNA sample from 0.1 to 0.2 millimolar, wherein the reaction condition composition does not require an intermediary step of reducing un-ligated circularizing oligonucleotide probes and is a single composition configured to conduct ligation and amplification of the at least one circularizing oligonucleotide probe simultaneously.