US 9,809,847 B2
Methods and compositions for detecting polynucleotides and fragments thereof
Sergei A. Kazakov, San Jose, CA (US); Catharina Casper-Lindley, Santa Cruz, CA (US); Anne Dallas, Santa Cruz, CA (US); Heini Ilves, Santa Cruz, CA (US); and Brian H. Johnston, Scotts Valley, CA (US)
Assigned to SOMAGENICS, INC., Santa Cruz, CA (US)
Appl. No. 15/318,640
Filed by SomaGenics, Inc., Santa Cruz, CA (US)
PCT Filed Jun. 19, 2015, PCT No. PCT/US2015/036761
§ 371(c)(1), (2) Date Dec. 13, 2016,
PCT Pub. No. WO2015/196120, PCT Pub. Date Dec. 23, 2015.
Claims priority of provisional application 62/014,569, filed on Jun. 19, 2014.
Prior Publication US 2017/0121762 A1, May 4, 2017
Int. Cl. C12Q 1/68 (2006.01)
CPC C12Q 1/6851 (2013.01) 21 Claims
1. A method for detecting a target polynucleotide having a length of about 10 to about 250 nucleotides, comprising:
a. hybridizing the target polynucleotide with a target specific oligonucleotide (TSO)
to produce a TSO-hybridized target polynucleotide, wherein the TSO comprises:
i. a first TSO region that hybridizes to a first target polynucleotide region;
ii. a second TSO region that hybridizes to a second target polynucleotide region; and
iii. a third TSO region that hybridizes to a third target polynucleotide region; wherein the second TSO region connects the first and third TSO regions; and wherein upon hybridization of the TSO to the target polynucleotide, the first and third of target polynucleotide regions become substrates for cleavage by a cleaving agent, and the second target polynucleotide region is not a substrate for cleavage by the cleaving agent;
b. cleaving the target polynucleotide at a first site within the first target polynucleotide region and a second site within the third target polynucleotide region to generate a TSO-hybridized processed target polynucleotide,
c. amplifying the TSO-hybridized processed target polynucleotide to produce an amplified processed polynucleotide, wherein amplifying the TSO-hybridized processed target polynucleotide comprises circularizing the processed target polynucleotide to produce a circular processed target polynucleotide and amplifying the circular processed target polynucleotide: and
d. detecting the amplified processed polynucleotide, thereby detecting the target polynucleotide.