Probe library construction

Patent metadata

Field	Value
Publication number	US-10240146-B2
Application number	US-201515329651-A
Country	US
Kind code	B2
Filing date	Jul 29, 2015
Priority date	Jul 30, 2014
Publication date	Mar 26, 2019
Grant date	Mar 26, 2019

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

The invention claimed is:

1. A method of producing DNA by amplifying a plurality of oligonucleotides, comprising:
amplifying, without substantial selective amplification, at least some of a plurality of oligonucleotides contained in a common liquid using real-time 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.

2. The method of claim 1 , wherein the plurality of oligonucleotides have an average length of between 10 and 200 nucleotides.

3. The method of claim 1 , wherein the plurality of oligonucleotides includes at least 10 unique oligonucleotide sequences.

4. The method of claim 1 , wherein the unique oligonucleotide sequences each comprise a first region that is identical in the unique oligonucleotide sequences, and a second region that is not identical in the unique oligonucleotide sequences.

5. The method of claim 1 , wherein amplifying at least some of the plurality of oligonucleotides comprises exposing a least some of the plurality of oligonucleotides to primer-containing sequences.

6. The method of claim 1 , wherein at least some of the amplified oligonucleotides contain a promoter.

7. The method of claim 1 , 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.

8. The method of claim 7 , comprising amplifying the first set of oligonucleotides but not the second set of oligonucleotides.

9. The method of claim 7 , wherein the plurality of oligonucleotides comprises at least 2 sets of oligonucleotides having distinguishable common index regions.

10. The method of claim 1 , 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.

11. The method of claim 1 , wherein transcribing at least some of the amplified oligonucleotides comprises exposing the amplified oligonucleotides to an RNA polymerase.

12. The method of claim 1 , 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.

13. The method of claim 1 , wherein reverse transcribing the RNA comprises exposing the RNA to a reverse transcriptase.

14. The method of claim 1 , wherein reverse transcribing the RNA to produce transcribed DNA occurs without first purifying the RNA from components used to produce the RNA.

15. The method of claim 1 , further comprising purifying the RNA from components used to produce the RNA prior to reverse transcribing the RNA to produce transcribed DNA.

16. The method of claim 1 , 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.

17. The method of claim 16 , wherein the sequence containing a transcription primer is incorporated into the transcribed DNA.

18. The method of claim 1 , wherein selectively degrading the RNA relative to the transcribed DNA comprises chemically reducing the RNA.

19. The method of claim 1 , wherein the transcribed DNA is substantially single-stranded.

20. The method of claim 1 , wherein each oligonucleotide of a subset of the oligonucleotides comprises an index portion that is identical.

21. The method of claim 1 , 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

• President And Fellows Harvard College
• 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

• C07H21/02

  with ribosyl as saccharide radical · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q2525/143

  incorporating a promoter sequence · CPC title