SPLIT OLIGONUCLEOTIDE PARTNER PROBES
Disclosed herein, inter alia, are polynucleotide probes, methods, and kits useful for amplifying and detecting target nucleic acids.
1 . A method of amplifying a complementarity determining regions (CDR) sequence, said method comprising:
a) hybridizing a first oligonucleotide to a first sequence of a target polynucleotide, and hybridizing a second oligonucleotide to a second sequence of said target polynucleotide, wherein the CDR sequence is between said first and second sequence;
b) extending the second oligonucleotide along the CDR sequence with a polymerase to generate a complementary sequence and ligating said complementary sequence to the first oligonucleotide;
c) ligating the first oligonucleotide to the second oligonucleotide, thereby generating a circular oligonucleotide; and
d) amplifying the circular oligonucleotide by extending an amplification primer hybridized to the circular oligonucleotide with a strand-displacing polymerase, thereby generating an extension product comprising multiple complements of the target polynucleotide sequence.
2 . The method of claim 1 , further comprising detecting the extension product of step (d).
3 . The method of claim 1 , further comprising sequencing the extension product of step (d).
4 . (canceled)
5 . The method of claim 3 , wherein sequencing comprises extending a sequencing primer by incorporating a labeled nucleotide, or labeled nucleotide analogue, and detecting the label to generate a signal for each incorporated nucleotide or nucleotide analogue, wherein the sequencing primer is hybridized to the extension product.
6 . The method of claim 1 , wherein the method comprises amplifying a target polynucleotide sequence of a cell in situ.
7 . (canceled)
8 . The method of claim 6 , wherein amplifying the circular oligonucleotide comprises incubating the circular oligonucleotide with the strand-displacing polymerase (a) for about 1 minute to about 2 hours, and/or (b) at a temperature of about 20° C. to about 50° C.
9 . The method of claim 8 , wherein incubation with the strand-displacing polymerase is at a temperature of about 35° C. to about 42° C.
10 .- 12 . (canceled)
13 . The method of claim 1 , wherein the circular oligonucleotide is about 100 to about 1000 nucleotides in length.
14 . (canceled)
15 . The method of claim 1 , wherein the first oligonucleotide and the second oligonucleotide each independently comprise a barcode sequence.
16 . The method of claim 15 , wherein the first oligonucleotide comprises from 5′ to 3′ a first hybridization sequence, a primer binding sequence, and a first barcode sequence, and wherein the second oligonucleotide comprises from 3′ to 5′ a second hybridization sequence and a second barcode sequence.
17 . The method of claim 15 , wherein each barcode sequence is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length.
18 . The method of claim 15 , wherein each barcode sequence is selected from a known set of barcode sequences.
19 . The method of claim 18 , wherein each of the known set of barcode sequences is associated with a hybridization sequence from a known set of hybridization sequences.
20 . The method of claim 16 , wherein the first barcode sequence is associated with the first hybridization sequence, and wherein the second barcode sequence is associated with the second hybridization sequence.
21 . The method of claim 18 , wherein barcodes in the known set of barcodes have a specified Hamming distance.
22 - 25 . (canceled)
26 . The method of claim 1 , wherein the target polynucleotide is RNA.
27 .- 30 . (canceled)
31 . The method of claim 1 , wherein step c) comprises hybridizing a splint oligonucleotide to both the first oligonucleotide and the second oligonucleotide, and ligating the first oligonucleotide and the second oligonucleotide.
32 . The method of claim 1 , wherein step c) comprises hybridizing a first ligation oligonucleotide to the first oligonucleotide and hybridizing a second ligation oligonucleotide to the second oligonucleotide, and ligating the first oligonucleotide and the second oligonucleotide together and ligating the first ligation oligonucleotide and the second ligation oligonucleotide together.
33 . The method of claim 1 , wherein the extension product comprises three or more copies of the circular oligonucleotide.
34 . The method of claim 1 , wherein the first oligonucleotide comprises a protelomerase recognition sequence and the second oligonucleotide comprises a complementary protelomerase recognition sequence.
35 .- 67 . (canceled)
68 . The method of claim 1 , wherein said CDR sequence is a CDR3 sequence.
69 . The method of claim 1 , wherein said first sequence comprises an IgH-V sequence and said second sequence comprises an IgH-J sequence.