US 12,258,637 B2
Compositions and methods for enhancing and/or predicting DNA amplification
Lars Peters, Portland, ME (US); Stephen A. Judice, Portland, ME (US); Daniel Shaffer, Portland, ME (US); and Breck Parker, Portland, ME (US)
Assigned to ENVIROLOGIX INC., Portland, ME (US)
Filed by ENVIROLOGIX INC., Portland, ME (US)
Filed on Oct. 6, 2022, as Appl. No. 17/938,585.
Application 17/938,585 is a continuation of application No. 15/306,032, granted, now 11,505,836, previously published as PCT/US2015/027074, filed on Apr. 22, 2015.
Claims priority of provisional application 61/982,784, filed on Apr. 22, 2014.
Prior Publication US 2023/0220495 A1, Jul. 13, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/68 (2018.01); C07H 21/04 (2006.01); C12Q 1/6844 (2018.01); C12Q 1/6848 (2018.01); C12Q 1/6853 (2018.01); C12Q 1/6876 (2018.01); C12Q 1/689 (2018.01); C12Q 1/6895 (2018.01)
CPC C12Q 1/6895 (2013.01) [C07H 21/04 (2013.01); C12Q 1/6844 (2013.01); C12Q 1/6848 (2013.01); C12Q 1/6853 (2013.01); C12Q 1/6876 (2013.01); C12Q 1/689 (2013.01); C12Q 2600/156 (2013.01)] 10 Claims
 
1. A method of designing a primer oligonucleotide, the primer oligonucleotide comprising from 5′ to 3′:
i) a first region comprising a self-complementary sequence comprising from 5′ to 3′ the reverse complement of a nicking enzyme recognition sequence, a palindromic sequence, and the nicking enzyme recognition sequence, and
ii) a second region at least 16 nucleotides long that specifically binds to a complementary region on a target nucleic acid molecule, wherein the second region of the primer oligonucleotide comprises two or more 2′ modified nucleotides, wherein the two or more 2′ modified nucleotides are selected from the group consisting of 2′-O—methyl, 2′-methoxyethoxy, 2′-fluoro, 2′-hydroxyl, 2′-alkyl, 2′-allyl, 2′-O-[2-(methylamino)-2-oxoethyl], 4′-CH2—O-2′-bridge, 4′-(CH2)2—O-2′-bridge, 2′-LNA, and 2′-O-(N-methylcarbamate) and those comprising base analogs, and wherein two or more 2′ modified nucleotides are contiguous;
the method comprising:
a) selecting the second region of said primer oligonucleotide, so that the ΔG for a double-stranded primer-target hybrid formed by the hybridization of the second region and the target nucleic acid molecule is at least 15 kcal/mol lower than the ΔG of a self-dimer comprising the second region of the primer; and
b) selecting a first region of said primer oligonucleotide, so that the ΔG for a homodimer formed by hybridization of the self-complementary first region of two primer oligonucleotide molecules is at least 15 kcal/mol lower than the ΔG of a self-dimer comprising the second region of the primer oligonucleotide.