METHODS AND SYSTEMS FOR SEQUENCING POLYNUCLEOTIDES
The present disclosure relates generally to detection on non-coding RNAS molecules in a sample or diagnosis of subject based upon detection or quantification of non-coding nucleic acid sequences in a sample, specifically to identify and use of molecular biomarkers for cancer including breast cancer.
1 . A method for sequencing a ribonucleic acid, comprising:
(a) providing the ribonucleic acid sequence or a functional fragment thereof;
(b) subjecting the ribonucleic acid or the functional fragment thereof to reverse transcription using a reverse transcriptase and a primer to generate a complementary deoxyribonucleic acid (cDNA) molecule, wherein the cDNA molecule comprises a sequence corresponding to a target sequence of the ribonucleic acid or the functional fragment thereof;
(c) amplifying the ribonucleic acid or the functional fragment thereof using one or more probes having different single stranded versus double stranded character; and
(d) sequencing the cDNA molecule or a derivative thereof.
2 . The method of claim 1 , wherein the ribonucleic acid sequence or the functional fragment thereof has a length of less than about 200 nucleotides.
3 . The method of claim 1 , wherein the ribonucleic acid sequence or the functional fragment thereof has a length of less between 50 and 100 nucleotides.
4 . The method of claim 1 , further comprising, after (b), amplifying the cDNA molecule.
5 . The method of claim 1 , wherein (c) comprises a Polymerase Chain Reaction (PCR).
6 . The method of claim 1 , wherein (c) occurs subsequent to (b).
7 . The method of claim 1 , wherein (c) comprises rolling circle amplification.
8 . The method of claim 1 , wherein (c) comprises a ligase chain reaction, multiplex ligatable probe amplification, in vitro transcription (IVT), strand displacement amplification, transcription-mediated amplification, or RNA (Eberwine) amplification.
9 . A reaction mixture, comprising:
a ribonucleic acid sequence or a functional fragment thereof;
one or more probes having different single stranded versus double stranded character for amplification of the ribonucleic acid or the functional fragment thereof;
a reverse transcriptase;
one or more primers; and
a complementary deoxyribonucleic acid molecule wherein the cDNA molecule comprises a sequence corresponding to a target sequence of the ribonucleic acid or the functional fragment thereof.
10 . The reaction mixture of claim 9 , wherein the ribonucleic acid sequence or the functional fragment thereof has a length of less than about 200 nucleotides.
11 . The reaction mixture of claim 9 , wherein the ribonucleic acid sequence or the functional fragment thereof has a length of less between 50 and 100 nucleotides.
12 . The reaction mixture of claim 9 , further comprising a single stranded oligonucleotide containing one or more universal primer sequences.
13 . The reaction mixture of claim 9 , further comprising one or more polyadenylated sequences.
14 . The reaction mixture of claim 9 , further comprising one or more adapter sequences.
15 . The reaction mixture of claim 9 , wherein the one or more primers comprises a primer complementary to a universal primer sequence.
16 . The reaction mixture of claim 9 , wherein the one or more primers comprises a poly(T) primer.
17 . The reaction mixture of claim 16 , wherein the one or more primers comprises a primer complementary to an adapter sequence of the one or more adapter sequences.
18 . The reaction mixture of claim 9 , wherein the one or more primers comprises an amplification primer that specifically binds to the ribonucleic acid or the functional fragment thereof.
19 . The reaction mixture of claim 9 , wherein the one or more primers comprises a reverse transcription primer.