Multimodal methods for simultaneous detection and quantification of multiple nucleic acids in a sample
View Patent ↗Described herein are approaches for the detection, identification, and/or quantification of target nucleic acids, including, but not limited to partially, substantially randomly, degraded target nucleic acids, in a biological sample, such as a formalin-fixed, paraffin-embedded (FFPE) sample. These approaches provide a means of detecting, identifying, and/or quantifying target nucleic acid molecules, including DNA and RNA molecules, further including RNAs of different classes, from the same sample, and in the same reaction, by using “expander oligonucleotides,” as the term is defined herein, to convert fragments of target nucleic acids into discretely sized DNA fragments, each with a chosen length characteristic for the target nucleic acid from which it is derived.
1. A method for producing a discretely sized surrogate marker indicative of the presence and/or quantity of each of a plurality of DNA and RNA target nucleic acid molecules present in a nucleic acid sample, the method comprising:
a) providing a nucleic acid sample comprising target DNA and target RNA nucleic acid molecules;
b) hybridizing a plurality of different reverse-transcription primers to the nucleic acid sample comprising target DNA and target RNA molecules of step (a), wherein each different reverse-transcription primer is specific for a different target RNA member of said plurality of target nucleic acid molecules;
c) extending the hybridized reverse-transcription primers of step (b) with an enzyme comprising reverse-transcriptase activity to produce a mixture comprising a population of cDNA molecules corresponding to the population of target RNA molecules hybridized in step (b), wherein the cDNA molecules corresponding to any given target RNA can differ in size;
d) hybridizing the nucleic acids existing in said mixture after step (c) with a plurality of different single-stranded expander oligonucleotides to produce a plurality of expander oligonucleotide:DNA hybrid molecules, wherein each said expander oligonucleotide (i) corresponds to one DNA or RNA member of the plurality of DNA and RNA target nucleic acid molecules, (ii) comprises a replica sequence that is substantially identical to a mirrored sequence within its corresponding target nucleic acid molecule, wherein, when the target nucleic acid molecule is an RNA molecule, said mirrored sequence is located closer to the 5′-end of said target RNA molecule than the sequence to which the reverse-transcription primers of step (b) hybridizes, and wherein T is substituted for U in the replica sequence;
e) extending the 3′ end of the expander oligonucleotide strand in each of the plurality of expander oligonucleotide:DNA hybrid molecules produced in step (d) using a template-dependent polymerase enzyme, the extension producing in the same reaction, for each target DNA and RNA nucleic acid molecule, a surrogate marker for the presence of each of the plurality of DNA and RNA target nucleic acid molecules present in the nucleic acid sample, wherein either: (i) each surrogate marker is of a different characteristic size relative to surrogate markers for other target nucleic acids; or amplification products of respective surrogate markers are of different characteristic sizes relative to other surrogate markers or amplification products thereof; and
f) amplifying at least a portion of the plurality of extended expander oligonucleotides by PCR amplification using a plurality of amplification primer pairs, thereby generating a plurality of extended expander oligonucleotide amplicons, wherein each said amplification primer pair comprises a forward amplification primer and a reverse amplification primer, wherein each said amplicon generated has a different length, and wherein each said amplicon generated provides a surrogate marker amplicon indicative of the presence and/or amount of one of said plurality of target nucleic acid molecules present in the nucleic acid sample.
2. The method of claim 1 , wherein said plurality of target nucleic acid molecules comprises target RNAs of different classes.
3. The method of claim 2 , wherein said target RNAs of different classes include two or more of mRNA, microRNA, siRNA, or hnRNA.
4. The method of claim 1 , wherein each said expander oligonucleotide further comprises a forward primer sequence 5′ of the replica sequence of the expander oligonucleotide.
5. The method of claim 1 , wherein each said expander oligonucleotide further comprises a spacer sequence, wherein the spacer sequence is immediately 5′ of the replica sequence of the expander oligonucleotide that is substantially identical to the mirrored sequence within the corresponding target nucleic acid molecule.
6. The method of claim 5 , wherein said spacer sequence within each member of the plurality of expander oligonucleotides is of a different, discrete length than said spacer sequence in all other members of said plurality of expander oligonucleotides.
7. The method of claim 1 , wherein said nucleic acid sample comprises partially, substantially randomly degraded target nucleic acids.
8. The method of claim 1 wherein each of the plurality of forward amplification primers used for amplification comprises a common 5′ tag sequence.
9. The method of claim 1 , wherein the plurality of forward amplification primers used for amplification are not all of the same length.
10. The method of claim 1 , wherein the plurality of reverse amplification primers used for amplification are not all of the same length.