Application No. 15/952,203
`Response to NFOA filed: January 21, 2020
`
`AMENDMENTS TO THE CLAIMS
`
`This listing of claims will replace all prior versions, and listings of claims in this application.
`
`Applicant reserves the right to pursue any subject matter of any canceled claims in this or any other
`
`appropriate patent application. Support for these claims is provided in the remarks following the
`
`listing of claims.
`
`Listing of the Claims
`
`1-1 14. (Cancelled)
`
`115.
`
`(Previously presented) A method for performing a primer extension reaction on
`
`RNA and DNA, comprising:
`
`a) providing a sample comprising a mixture of single-stranded DNA and single-
`
`stranded RNA,
`
`b) attaching a first adapter to said single-stranded DNA,
`
`0) attaching a second adapter to said single-stranded RNA,
`
`d) annealing a first primer to said first adapter and annealing a second primer to said
`
`second adapter,
`
`e) extending said annealed first primer on said single-stranded DNA to form double-
`
`stranded DNA, and
`
`f) extending said annealed second primer on said single-stranded RNA to form a
`
`double-stranded DNA-RNA hybrid.
`
`116.
`
`(Previously presented) The method of claim 115, wherein said attaching said first
`
`adapter comprises ligating said first adapter to a 3’ end of said single-stranded DNA.
`
`117.
`
`(Previously presented) The method of claim 116, wherein said ligating said first
`
`adapter is performed by a ligase selected from the group consisting of CircLigase II, Thermostable
`
`App-DNA/RNA ligase, T4 RNA ligase 1, T4 RNA Ligase 2 truncated, and any combination thereof.
`
`118.
`
`(Previously presented) The method of claim 115, wherein said attaching said second
`
`adapter comprises ligating said second adapter to a 3’ end of said single-stranded RNA.
`
`119.
`
`(Previously presented) The method of claim 118, wherein said ligating said second
`
`adapter is performed using an RNA ligase.
`
`-2-
`
`Atty. Docket No. 47697-709201
`
`

`

`Application No. 15/952,203
`Response to NFOA filed: January 21, 2020
`
`120.
`
`(Previously presented) The method of claim 118, wherein said ligating said second
`
`adapter is performed using T4 RNA ligase 2 or T4 DNA ligase.
`
`121.
`
`(Previously presented) The method of claim 115, wherein said single-stranded DNA
`
`is cell-free DNA.
`
`122.
`
`(Previously presented) The method of claim 115, wherein said sample is selected
`
`from the group consisting of blood, plasma, serum, cerebrospinal fluid, synovial fluid, bronchio-
`
`alveolar lavage, urine, stool, saliva, nasal swab, and any combination thereof.
`
`123.
`
`(Previously presented) The method of claim 115, wherein said extending said
`
`annealed first primer on said single-stranded DNA is performed by a DNA polymerase.
`
`124.
`
`(Previously presented) The method of claim 115, wherein said extending said
`
`annealed first primer on said single-stranded DNA is performed by Bst 2.0 DNA polymerase.
`
`125.
`
`(Currently amended) The method of claim 115, wherein said extending said annealed
`
`second primer on said single-stranded RNA is performed by a pel-ymerase—seleeted—frem—Bfleney
`
`
`
`.1.
`
`--
`
`-.
`
`.
`
`-.
`
`U U
`
`.
`
`-
`
`.-
`
`-:. -
`
`.-=..*.H’
`
`-
`
`reversetranscriptase.
`
`126.
`
`(Previously presented) The method of claim 115, further comprising adding at least
`
`one non-templated nucleotide to a first primer extension strand.
`
`127.
`
`(Previously presented) The method of claim 126, wherein said at least one non-
`
`templated nucleotide is a deoxycytidine.
`
`128.
`
`(Previously presented) The method of claim 126, wherein said at least one non-
`
`templated nucleotide is added to a 3’ end.
`
`129.
`
`(Previously presented) The method of claim 126, wherein said at least one non-
`
`templated nucleotide is up to eight nucleotides.
`
`130.
`
`(Previously presented) The method of claim 126, wherein said at least one non-
`
`templated nucleotide is three, four, or five non-templated nucleotides.
`
`131.
`
`(Previously presented) The method of claim 126, wherein said at least one non-
`
`templated nucleotide is one non-templated nucleotide.
`
`132.
`
`(Previously presented) The method of claim 126, wherein said at least one non-
`
`templated nucleotide forms a first overhang.
`
`-3-
`
`Atty. Docket No. 47697-709201
`
`

`

`Application No. 15/952,203
`Response to NFOA filed: January 21, 2020
`
`133.
`
`(Previously presented) The method of claim 132, further comprising adding at least
`
`one second non-templated nucleotide to a second primer extension strand forming a second
`
`overhang.
`
`134.
`
`(Previously presented) The method of claim 133, further comprising hybridizing a
`
`third adapter to said first overhang and a fourth adapter to said second overhang.
`
`135.
`
`(Previously presented) The method of claim 134, further comprising sequencing said
`
`third adapter and said fourth adapter and sequences attached to said third adapter and said fourth
`
`adapter.
`
`136.
`
`(Previously presented) The method of claim 134, further comprising (i) identifying
`
`sequences associated with said third adapter as originating from said DNA in said mixture of single-
`
`stranded DNA and single-stranded RNA and (ii) identifying sequences associated with said fourth
`
`adapter as originating from said RNA in said mixture of single-stranded DNA and single-stranded
`
`RNA.
`
`137. — 151. (Cancelled)
`
`[Remainder ofpage intentionally left blank]
`
`-4-
`
`Atty. Docket No. 47697-709201
`
`