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  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6844—Nucleic acid amplification reactions
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6869—Methods for sequencing

Definitions

• the DNA polymerase is selected from the group consisting of: Taq polymerase, Pheonix Taq polymerase, Phusion polymerase, T4 polymerase, T7 polymerase, Klenovv fragment, lenovv exo-, phi29 polymerase, VeraSeq ULtra polymerase, and EnzScript.
• the RNA polymerase is selected from the group consisting of: T3 polymerase, T7 polymerase, and SP6 polymerase.
• the at least one oligonucleotide further comprises at least one of a barcode sequence, an index sequence and an adapter sequence.
• the container is a chamber of a multichamber cartridge.
• step 103 the remaining DNA molecule is contacted by one or more target-specific oligonucleotides such that a target-specific oligonucleotide hybridizes to a region of the DNA and is extended to synthesize a complementary DNA strand.
• this reaction is performed by Phoenix DNA polymerase.
• this reaction is performed by a dual function reverse transcriptase that also has DNA polymerase activity ⁇ e.g. , AMV reverse transcriptase enzyme).
• RNA polymerase transcribes, using the RNA polymerase promoter contained within the common sequence, an RNA molecule complementary to the DNA template.
• steps 101- 104 can be repeated through multiple cycles of each of which begins with the complementary RNA molecule resulting from step 104 serving as the template at step 101.
• the transcribed RNA may be subsequently purified in step 105. .
• step 106 the purified RNA containing the 5 ' common sequence is then contacted by one or more target-specific oligonucleotides.
• Target-specific oligonucleotide # 1 hybridizes with the complementary RNA at a target sequence and primes a template-dependent reverse transcriptase reaction producing a complementary DNA strand.
• step 107 the RNA template is enzyiTiatically degraded ⁇ e.g., through the RNase activity) from the complementary hybrid RNA-DNA molecule of RNaseH.
• the remaining DNA molecule is contacted by an oligonucleotide containing a sequence complementary to the common sequence present on the 3 ' end of the DNA molecule as well as an additional sequence that may contain any one or more sequences including barcode, index, and adapter sequences.
• the oligonucleotide hybridizes and is extended to produce a complementary DNA strand that is purified in step 125.
• the resulting DNA molecule is double stranded and contains the target sequence and its adjacent region flanked by additional sequences that contain adapter sequences for the appropriate sequencing platform.
• step 208 the remaining DNA molecu le is contacted by an oligonucleotide containing a sequence encoding an RNA polymerase promoter 5 ' to a sequence complementary to the common sequence present on the 3 ' end of the DNA molecule.
• the oligonucleotide is extended to produce a complementary DNA strand.
• RNA polymerase utilizes the RNA polymerase promoter to transcribe a complementary RNA molecule.
• Steps 206-209 are repeated through multiple cycles each of which begins with the complementary RNA molecule resulting from step 209 serving as the template at step 206.
• RNA molecules from reaction 209 may also be purified as in step 210 in preparation for subsequent steps.
• steps 201-209 are performed consecutively in a single reaction tube.
• steps 201-209 are performed as isothermal reactions.
• FIG. 4 illustrates an exemplary process for preparing nucleic acids having a target region with a 5' and/or 3 ' adjacent region.
• an initial RNA is obtained or provided as a template molecule.
• the RNA template is exposed to a plurality of ol igonucleotides that share a common sequence (common sequence # 1 ) that is 5 ' to different hybridization sequences.
• step 41 1 the purified RNA by one or more target-specific oligonucleotide containing additional sequences 5 ' to the target-specific sequence.
• additional sequences include but are not limited to barcode, index, and/or adapter sequences.
• the target- specific oligonucleotide hybridizes with the complementary RNA at a target sequence and primes a template-dependent reverse transcriptase reaction producing a complementary DNA strand.
• the RNA template is enzymatically degraded (e.g., RNase H activity) from the complementary hybrid RNA-DNA molecule.
• RNA purification was performed as above, this time eluting the RNA in 6 i 10 mM Tris-HCl pH8.3 Elution Buffer.
• Example 2 An isothermal method using reverse transcriptase with tagged randomers and targeted second strand oligonucleotides to a mplify 5'fusion events
• Example 3 An isothermal method to exponentially amplify target sequences beginning with genomic DNA.

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• 2015
  • 2015-01-26 CA CA2938141A patent/CA2938141A1/en not_active Abandoned
  • 2015-01-26 CN CN201580016111.4A patent/CN107075566B/zh not_active Expired - Fee Related
  • 2015-01-26 EP EP15739782.9A patent/EP3099819A4/en not_active Withdrawn
  • 2015-01-26 AU AU2015209103A patent/AU2015209103B2/en not_active Ceased
  • 2015-01-26 US US14/605,030 patent/US20150337364A1/en not_active Abandoned
  • 2015-01-26 CN CN202110571327.4A patent/CN113388664A/zh not_active Withdrawn
  • 2015-01-26 JP JP2016549245A patent/JP2017504346A/ja active Pending
  • 2015-01-26 WO PCT/US2015/012842 patent/WO2015112949A2/en not_active Ceased
• 2021
  • 2021-03-26 JP JP2021053293A patent/JP2021094038A/ja not_active Withdrawn
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