Systems and methods for determining nucleic acids

Abstract

The present invention generally relates to systems and methods for imaging or determining nucleic acids, for instance, within cells. In some embodiments, the transcriptome of a cell may be determined. Certain embodiments are directed to determining nucleic acids, such as mRNA, within cells at relatively high resolutions. In some embodiments, a plurality of nucleic acid probes may be applied to a sample, and their binding within the sample determined, e.g., using fluorescence, to determine locations of the nucleic acid probes within the sample. In some embodiments, codewords may be based on the binding of the plurality of nucleic acid probes, and in some cases, the codewords may define an error-correcting code to reduce or prevent misidentification of the nucleic acids. In certain cases, a relatively large number of different targets may be identified using a relatively small number of labels, e.g., by using various combinatorial approaches.

1 - 176 . (canceled) 
     
     
         177 . A method for determining a plurality of different nucleic acid targets within a sample by in situ hybridization, comprising:
 (a) contacting a sample comprising a plurality of different nucleic acid targets with a plurality of primary nucleic acid probes, wherein each primary nucleic acid probe comprises (i) a target sequence and (ii) one or more read sequences, wherein the target sequence hybridizes to the nucleic acid target in the sample to form a primary probe-hybridized complex;   (b) contacting the sample to a second round of hybridization using a plurality of secondary nucleic acid probes wherein the secondary nucleic acid probes comprise a first portion that hybridizes to a subset of the read sequences of the primary nucleic acid probe and a second portion comprising a fluorescent label to form a secondary probe-hybridized complex;   (c) detecting a fluorescent signal from each of the secondary probe-hybridized complexes;   (d) removing the secondary nucleic acid probes or inactivating the fluorescent signal from each of the secondary probe-hybridized complexes; and   (e) repeating steps (b) through (d) with multiple sets of differing secondary nucleic acid probes to generate a pattern of binding of the secondary nucleic acid probes;
 wherein the pattern of binding of the secondary nucleic acid probes is converted to a codeword, wherein the codeword determines the identity of the nucleic acid targets, and wherein the codeword provides an error correction system. 
   
     
     
         178 . The method of  claim 177 , wherein the target sequence of the plurality of primary nucleic acid probes comprises an average length of between 10 and 200 nucleotides. 
     
     
         179 . The method of  claim 177 , wherein the nucleic acid target is RNA. 
     
     
         180 . The method of  claim 179 , wherein the method comprises determining the transcriptome of a cell. 
     
     
         181 . The method of  claim 180 , wherein at least 25% of the transcriptome is determined. 
     
     
         182 . The method of  claim 177 , wherein the plurality of primary nucleic acid probes comprises at least 10 different primary nucleic acid probes. 
     
     
         183 . The method of  claim 177 , wherein the plurality of primary nucleic acid probes comprises at least 8 read sequences. 
     
     
         184 . The method of  claim 177 , wherein each set of the plurality of secondary nucleic acid probes hybridizes to a subset of the read sequences. 
     
     
         185 . The method of  claim 177 , wherein the one or more read sequences have an average length of at least 15 nucleotides. 
     
     
         186 . The method of  claim 177 , wherein the fluorescent signal is inactivated by photobleaching. 
     
     
         187 . The method of  claim 177 , wherein the fluorescent signal is inactivated by chemically or enzymatically cleaving the fluorescent label from the readout probe-hybridized complexes. 
     
     
         188 . The method of  claim 177 , wherein the fluorescent signal is detected using a fluorescence imaging technique. 
     
     
         189 . The method of  claim 177 , wherein the one or more read sequences are distributed on the plurality of primary nucleic acid probes so as to define an error-correcting code. 
     
     
         190 . The method of  claim 177 , wherein if the pattern of binding of the secondary nucleic acid probes identifies a codeword that does not match a valid codeword, error correction is applied to the codeword to form a valid codeword. 
     
     
         191 . The method of  claim 177 , wherein the error correction system comprises a Hamming system, a Golay code, or an extended Hamming system. 
     
     
         192 . The method of  claim 177 , wherein the error correction system identifies the location of an error. 
     
     
         193 . The method of  claim 177 , wherein the error correction system identifies a single bit error. 
     
     
         194 . The method of  claim 177 , wherein the error correction system identifies a two bit error. 
     
     
         195 . The method of  claim 177 , wherein the error correction system corrects a single bit error. 
     
     
         196 . The method of  claim 191 , wherein the plurality of primary nucleic acid probes defines a code space with a Hamming distance of at least 2. 
     
     
         197 . The method of  claim 177 , wherein each codeword comprises at least a 14 bit code. 
     
     
         198 . The method of  claim 177 , wherein the binding pattern of secondary nucleic acid probes is used to determine the location of the primary nucleic acid probes within the sample. 
     
     
         199 . The method of  claim 177 , wherein a given set of differing secondary nucleic acid probes has differing fluorescent labels. 
     
     
         200 . The method of  claim 177 , wherein multiple sets of differing secondary nucleic acid probes have the same fluorescent label. 
     
     
         201 . The method of  claim 177 , wherein the presence of the fluorescent signal is assigned a value in the codeword. 
     
     
         202 . The method of  claim 177 , wherein the absence of the fluorescent signal is assigned a value in the codeword. 
     
     
         203 . The method of  claim 177 , wherein the determining of the plurality of different nucleic acid targets within the sample is qualitative and/or quantitative. 
     
     
         204 . The method of  claim 177 , wherein the determination of the plurality of different nucleic acid targets within the sample is spatial. 
     
     
         205 . The method of  claim 177 , wherein the position of the primary nucleic acid probe within the sample is determined in at least 2 dimensions. 
     
     
         206 . The method of  claim 177 , wherein the position of the primary nucleic acid probe within the sample is determined in at least 3 dimensions.