OLIGONUCLEOTIDE PROBES AND USES THEREOF
Methods and compositions are provided for oligonucleotide probes and oligonucleotide probe libraries that recognize targets of interest. The targets include circulating biomarkers such as microvesicles, including those derived from various diseases.
1 - 69 . (canceled)
70 . A method of generating a single-stranded DNA (ssDNA) molecule comprising:
(a) providing a mixture comprising:
(i) a nucleic acid molecule comprising:
(1) a 5′ leader region comprising a lengthener region, a terminator region, and a forward primer region;
(2) a variable region positioned 3′ of the leader region; and
(3) a tail region positioned 3′ of the variable region and comprising a reverse primer region; and
(ii) forward and reverse primers configured to amplify the nucleic acid molecule from the forward primer region and reverse primer region, respectively; and
(b) performing asymmetric polymerase chain reaction (PCR) on the mixture in a) to favorably amplify the reverse strand of the nucleic acid molecule, wherein the forward and reverse primers in the mixture are at a ratio of between about 1:20-1:50 (F/R) in favor of the reverse primers; thereby generating the ssDNA molecule.
71 . (canceled)
72 . The method of claim 70 , wherein the ratio is between about 1:37.5 (F/R) in favor of the reverse primers.
73 . The method of claim 70 , further comprising isolating the amplified reverse strand of the nucleic acid molecule on a native gel.
74 . The method of claim 70 , further comprising:
(c) denaturing the amplified nucleic acid molecules from b); and
(d) isolating the denatured reverse strand of the nucleic acid molecules from c).
75 . The method of claim 74 , wherein the denatured reverse strand of the nucleic acid molecules is isolated on a denaturing gel.
76 . The method of claim 70 , wherein the mixture in a) further comprises at least one of an enrichment buffer, non-target molecules, proteins, microvesicles, and polyethylene glycol.
77 - 79 . (canceled)
80 . The method of claim 70 , wherein the lengthener region comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 30, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides.
81 . The method of claim 70 , wherein the lengthener region comprises a poly-A sequence.
82 . The method of claim 70 , wherein the terminator region comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 30, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides.
83 . The method of claim 70 , wherein the terminator region comprises a non-nucleotide terminator.
84 . The method of claim 83 , wherein the non-nucleotide terminator comprises triethylene glycol.
85 . The method of claim 70 , wherein the forward primer region comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 30, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides.
86 . The method of claim 70 , wherein the variable region comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 30, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides.
87 . The method of claim 86 , wherein the variable region binds a target molecule or complex through non-Watson-Crick base pairing.
88 . The method of claim 70 , wherein the reverse primer region comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 30, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides.