Application No. 15/952,203
`Response to NFOA filed: January 21, 2020
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`AMENDMENTS TO THE CLAIMS
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`This listing of claims will replace all prior versions, and listings of claims in this application.
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`Applicant reserves the right to pursue any subject matter of any canceled claims in this or any other
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`appropriate patent application. Support for these claims is provided in the remarks following the
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`listing of claims.
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`Listing of the Claims
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`1-1 14. (Cancelled)
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`115.
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`(Previously presented) A method for performing a primer extension reaction on
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`RNA and DNA, comprising:
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`a) providing a sample comprising a mixture of single-stranded DNA and single-
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`stranded RNA,
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`b) attaching a first adapter to said single-stranded DNA,
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`0) attaching a second adapter to said single-stranded RNA,
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`d) annealing a first primer to said first adapter and annealing a second primer to said
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`second adapter,
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`e) extending said annealed first primer on said single-stranded DNA to form double-
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`stranded DNA, and
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`f) extending said annealed second primer on said single-stranded RNA to form a
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`double-stranded DNA-RNA hybrid.
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`116.
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`(Previously presented) The method of claim 115, wherein said attaching said first
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`adapter comprises ligating said first adapter to a 3’ end of said single-stranded DNA.
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`117.
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`(Previously presented) The method of claim 116, wherein said ligating said first
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`adapter is performed by a ligase selected from the group consisting of CircLigase II, Thermostable
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`App-DNA/RNA ligase, T4 RNA ligase 1, T4 RNA Ligase 2 truncated, and any combination thereof.
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`118.
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`(Previously presented) The method of claim 115, wherein said attaching said second
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`adapter comprises ligating said second adapter to a 3’ end of said single-stranded RNA.
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`119.
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`(Previously presented) The method of claim 118, wherein said ligating said second
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`adapter is performed using an RNA ligase.
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`-2-
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`Atty. Docket No. 47697-709201
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`
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`Application No. 15/952,203
`Response to NFOA filed: January 21, 2020
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`120.
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`(Previously presented) The method of claim 118, wherein said ligating said second
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`adapter is performed using T4 RNA ligase 2 or T4 DNA ligase.
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`121.
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`(Previously presented) The method of claim 115, wherein said single-stranded DNA
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`is cell-free DNA.
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`122.
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`(Previously presented) The method of claim 115, wherein said sample is selected
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`from the group consisting of blood, plasma, serum, cerebrospinal fluid, synovial fluid, bronchio-
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`alveolar lavage, urine, stool, saliva, nasal swab, and any combination thereof.
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`123.
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`(Previously presented) The method of claim 115, wherein said extending said
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`annealed first primer on said single-stranded DNA is performed by a DNA polymerase.
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`124.
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`(Previously presented) The method of claim 115, wherein said extending said
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`annealed first primer on said single-stranded DNA is performed by Bst 2.0 DNA polymerase.
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`125.
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`(Currently amended) The method of claim 115, wherein said extending said annealed
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`second primer on said single-stranded RNA is performed by a pel-ymerase—seleeted—frem—Bfleney
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`
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`.1.
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`.-
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`.-=..*.H’
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`-
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`reversetranscriptase.
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`126.
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`(Previously presented) The method of claim 115, further comprising adding at least
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`one non-templated nucleotide to a first primer extension strand.
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`127.
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`(Previously presented) The method of claim 126, wherein said at least one non-
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`templated nucleotide is a deoxycytidine.
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`128.
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`(Previously presented) The method of claim 126, wherein said at least one non-
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`templated nucleotide is added to a 3’ end.
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`129.
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`(Previously presented) The method of claim 126, wherein said at least one non-
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`templated nucleotide is up to eight nucleotides.
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`130.
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`(Previously presented) The method of claim 126, wherein said at least one non-
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`templated nucleotide is three, four, or five non-templated nucleotides.
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`131.
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`(Previously presented) The method of claim 126, wherein said at least one non-
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`templated nucleotide is one non-templated nucleotide.
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`132.
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`(Previously presented) The method of claim 126, wherein said at least one non-
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`templated nucleotide forms a first overhang.
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`-3-
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`Atty. Docket No. 47697-709201
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`
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`Application No. 15/952,203
`Response to NFOA filed: January 21, 2020
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`133.
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`(Previously presented) The method of claim 132, further comprising adding at least
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`one second non-templated nucleotide to a second primer extension strand forming a second
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`overhang.
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`134.
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`(Previously presented) The method of claim 133, further comprising hybridizing a
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`third adapter to said first overhang and a fourth adapter to said second overhang.
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`135.
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`(Previously presented) The method of claim 134, further comprising sequencing said
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`third adapter and said fourth adapter and sequences attached to said third adapter and said fourth
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`adapter.
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`136.
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`(Previously presented) The method of claim 134, further comprising (i) identifying
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`sequences associated with said third adapter as originating from said DNA in said mixture of single-
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`stranded DNA and single-stranded RNA and (ii) identifying sequences associated with said fourth
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`adapter as originating from said RNA in said mixture of single-stranded DNA and single-stranded
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`RNA.
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`137. — 151. (Cancelled)
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`[Remainder ofpage intentionally left blank]
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`-4-
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`Atty. Docket No. 47697-709201
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