Compositions and Methods for Intramolecular Nucleic Acid Rearrangement
Aspects of the present invention are drawn to processes for moving a region of interest in a polynucleotide from a first position to a second position with regard to a domain within the polynucleotide, also referred to as a “reflex method”. In certain embodiments, the reflex method results in moving a region of interest into functional proximity to specific domain elements present in the polynucleotide (e.g., primer sites and/or MID). Compositions, kits and systems that find use in carrying out the reflex processes described herein are also provided.
1 - 20 . (canceled)
21 . A method of analyzing nucleic acids, comprising:
providing a plurality of nucleic acids from a single cell source;
tagging the plurality of nucleic acids from the single cell source with multiplex identifier (MID) sequences; and
determining a nucleotide sequence of the plurality of nucleic acids and attached MID sequences.
22 . The method of claim 21 , wherein the MID sequences uniquely identify the single cell source.
23 . The method of claim 21 , wherein the MID sequences uniquely identify each of a plurality of nucleic acid molecules from the single cell source.
24 . The method of claim 21 , further comprising amplifying the plurality of nucleic acids and attached MID sequences before determining the nucleotide sequence of the plurality of nucleic acids and attached MID sequences.
25 . The method of claim 21 , wherein the determining step comprises sequencing the plurality of nucleic acids and attached MID sequences.
26 . The method of claim 21 , wherein the plurality of nucleic acids is selected from DNA and RNA
27 . The method of claim 26 , wherein the plurality of nucleic acids comprises mRNA.
28 . The method of claim 23 , further comprising identifying a number of the plurality of nucleic acids tagged with a unique MID, and determining a number of the plurality of nucleic acid molecules that were within a single cell source from a number of unique MID sequences identified.
29 . The method of claim 21 , wherein the tagging step comprises ligating a sequence comprising the MID sequences to the plurality nucleic acids.
30 . The method of claim 21 , wherein the tagging step comprises performing PCR or linear amplification of the plurality of nucleic acids using an adapter sequence comprising the MID sequence and an amplification primer.
31 . The method of claim 21 , wherein:
the providing step comprises providing a plurality of nucleic acids from each of a plurality of single cell sources;
the attaching step comprises attaching different MID sequences to the nucleic acids from each of the plurality of single cell sources; and
wherein nucleic acid sequences are attributed to different individual cell sources based upon the MID sequences attached in the attaching step.
32 . The method of claim 21 , wherein the MID sequences further comprise a sequencing primer sequence.
33 . The method of claim 21 , wherein the MID sequences are from 2 to 100 bases in length.
34 . The method of claim 31 , wherein the plurality of different MID sequences comprises at least 100 different MID sequences.
35 . A method of analyzing nucleic acids, comprising:
providing a plurality of nucleic acids from a single cell source;
tagging the plurality of nucleic acids from the single cell source with multiplex identifier (MID) sequences, wherein each of the plurality of molecules is tagged with a unique MID sequence; and
analyzing the plurality of nucleic acids and attached MID sequences.
36 . The method of claim 35 , wherein the analyzing step comprises sequencing the plurality of nucleic acids and attached MID sequences.
37 . A method of analyzing nucleic acids, comprising:
providing a plurality of nucleic acids from a single cell source;
tagging the plurality of nucleic acids from the single cell source with multiplex identifier (MID) sequences, wherein each of the plurality of molecules is tagged with a unique MID sequence;
amplifying the plurality of nucleic acids and associated MID sequences to provide amplified nucleic acids; and
determining a nucleotide sequence of amplified nucleic acids.
38 . The method of claim 37 , wherein the amplifying step comprises performing a polymerase chain reaction amplification.
39 . The method of claim 37 , wherein the amplifying step comprises a linear amplification step.