US 11,987,836 B2
Method for nucleic acid analysis directly from an unpurified biological sample
Ryan Charles Heller, Niskayuna, NY (US); Nichole Lea Wood, Niskayuna, NY (US); Robert Scott Duthie, Niskayuna, NY (US); John Richard Nelson, Niskayuna, NY (US); Wei Gao, Niskayuna, NY (US); Michael James Rishel, Niskayuna, NY (US); and Klaus Gustav Hentrich, Cardiff (GB)
Assigned to GLOBAL LIFE SCIENCES SOLUTIONS USA LLC, Marlborough, MA (US)
Appl. No. 15/510,331
Filed by Global Life Sciences Solutions USA LLC, Marlborough, MA (US)
PCT Filed Sep. 17, 2015, PCT No. PCT/US2015/050760
§ 371(c)(1), (2) Date Mar. 10, 2017,
PCT Pub. No. WO2016/053638, PCT Pub. Date Apr. 7, 2016.
Claims priority of provisional application 62/057,401, filed on Sep. 30, 2014.
Prior Publication US 2017/0298415 A1, Oct. 19, 2017
Int. Cl. C12Q 1/6806 (2018.01); C12Q 1/6844 (2018.01); C12Q 1/6858 (2018.01); C12Q 1/6869 (2018.01)
CPC C12Q 1/6806 (2013.01) [C12Q 1/6844 (2013.01); C12Q 1/6858 (2013.01); C12Q 1/6869 (2013.01)] 7 Claims
 
1. A method for generating single-stranded DNA circles from a biological sample, the method comprising:
A) treating the biological sample with an extractant to release nucleic acids, thereby forming a sample mixture, and subjecting the sample mixture to a drying step on a solid matrix, wherein the solid matrix comprises glass fiber filters or quartz fiber filters (QMA), and the solid matrix is impregnated with a chelating agent, an anionic surfactant, or a combination thereof;
B) neutralizing the extractant, wherein the extractant is neutralized by adding a sequestrant comprising a proteinase K inhibitor;
C) denaturing the released nucleic acids to generate single-stranded DNA;
D) contacting the single-stranded DNA with a ligase that is capable of template-independent, intramolecular ligation of a single-stranded DNA sequence to generate the single-stranded DNA circles; and
E) amplifying the single-stranded DNA circles via rolling circle amplification, wherein the single-stranded DNA circles are amplified with primers comprising one or more nucleotide analogues selected from a locked nucleic acid, a 2-Amino-dA, or a 2-Thio-dT,
wherein the quantity of DNA extracted from the biological sample is limiting or of low quality, and wherein all the steps of the method are performed without any intermediate nucleic acid isolation or nucleic acid purification.