Probe library construction

Patent metadata

Field	Value
Publication number	US-2019233812-A1
Application number	US-201916253919-A
Country	US
Kind code	A1
Filing date	Jan 22, 2019
Priority date	Jul 30, 2014
Publication date	Aug 1, 2019
Grant date	—

Abstract

The present invention generally relates to systems and methods for producing nucleic acids. In some aspects, relatively large quantities of oligonucleotides can be produced, and in some cases, the oligonucleotides may have a variety of different sequences and/or lengths. For instance, a relatively small quantity of oligonucleotides may be amplified to produce a large amount of nucleotides. In one set of embodiments, oligonucleotides may be amplified using PCR, then transcribed to produce RNA. The RNA may then be reverse transcribed to produce DNA, and optionally, the RNA may be selectively degraded or removed, relative to the DNA. In one set of embodiments, the oligonucleotides may be chemically modified. These modifications may include, but are not limited, to the adding of fluorescent dyes or other signaling entities.

First claim

1 - 72 . (canceled)

73 . A method, comprising:
simultaneously amplifying at least some of a plurality of oligonucleotides in a common solution using PCR to produce amplified oligonucleotides; transcribing in vitro at least some of the amplified oligonucleotides to produce RNA; reverse transcribing the RNA to produce transcribed DNA; and selectively degrading the RNA relative to the transcribed DNA.

74 . The method of claim 73 , wherein the plurality of oligonucleotides have an average length of between 10 and 200 nucleotides.

75 . The method of claim 73 , wherein the plurality of oligonucleotides includes at least 10 unique oligonucleotide sequences.

76 - 79 . (canceled)

80 . The method of claim 73 , wherein amplifying at least some of the plurality of oligonucleotides comprises exposing at least some of the plurality of oligonucleotides to primer-containing sequences.

81 . (canceled)

82 . The method of claim 80 , wherein at least some of the amplified oligonucleotides contain a promoter.

83 - 85 . (canceled)

86 . The method of claim 73 , wherein at least some of the plurality of oligonucleotides comprise at least a first set of oligonucleotides having a first common index region, and a second set of oligonucleotides having a second common index region distinguishable from the first common index region.

87 . The method of claim 86 , comprising amplifying the first set of oligonucleotides but not the second set of oligonucleotides.

88 . The method of claim 86 , wherein the plurality of oligonucleotides comprises at least 2 sets of oligonucleotides having distinguishable common index regions.

89 - 95 . (canceled)

96 . The method of claim 73 , wherein transcribing at least some of the amplified oligonucleotides comprises a mass of RNA that is at least 100-fold greater than the mass of amplified oligonucleotides.

97 . The method of claim 73 , wherein transcribing at least some of the amplified oligonucleotides comprises exposing the amplified oligonucleotides to an RNA polymerase.

98 - 102 . (canceled)

103 . The method of claim 73 , wherein transcribing at least some of amplified oligonucleotides to produce RNA comprises producing, on average, at least 10 RNA copies of each of the amplified oligonucleotides.

104 - 106 . (canceled)

107 . The method of claim 73 , wherein reverse transcribing the RNA comprises exposing the RNA to a reverse transcriptase.

108 . (canceled)

109 . The method of claim 73 , wherein reverse transcribing the RNA to produce transcribed DNA occurs without first purifying the RNA from components used to produce the RNA.

110 . The method of claim 73 , further comprising purifying the RNA from components used to produce the RNA prior to reverse transcribing the RNA to produce transcribed DNA.

111 . The method of claim 73 , wherein reverse transcribing the RNA to produce transcribed DNA comprises reverse transcribing the RNA to produce transcribed DNA using a sequence containing a transcription primer.

112 . The method of claim 111 , wherein the sequence containing a transcription primer is incorporated into the transcribed DNA.

113 - 122 . (canceled)

123 . The method of claim 73 , wherein selectively degrading the RNA relative to the transcribed DNA comprises chemically reducing the RNA.

124 - 126 . (canceled)

127 . The method of claim 73 , wherein the transcribed DNA is substantially single-stranded.

128 - 140 . (canceled)

141 . The method of any one of claim 73 , wherein each oligonucleotide of a subset of the oligonucleotides comprises an index portion that is identical.

142 . (canceled)

143 . The method of claim 73 , wherein the plurality of oligonucleotides has a distribution of lengths such that no more than 10% of the oligonucleotides has a length that is less than 80% or greater than 120% of the overall average length of the plurality of nucleotides.

Assignees

• Harvard College

Inventors

• Zhuang Xiaowei
• Moffitt Jeffrey R
• Boettiger Alistair

Classifications

• G06N7/01

  Probabilistic graphical models, e.g. probabilistic networks · CPC title

• G16C20/10

  Analysis or design of chemical reactions, syntheses or processes · CPC title

• C12N15/1093

  General methods of preparing gene libraries, not provided for in other subgroups · CPC title

• C12Q1/6816

  characterised by the detection means (C12Q1/6804 takes precedence) · CPC title

• C12N15/1065 Primary

  Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags · CPC title