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Methods of sequencing nucleic acids in mixtures and compositions related thereto

A technology of mixtures and compositions, applied in biochemical equipment and methods, measurement/testing of microorganisms, recombinant DNA technology, etc., can solve the problems of indistinguishable transcripts and incomplete sequencing

Active Publication Date: 2015-12-02
EMORY UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Individual transcripts are indistinguishable, or cannot be fully sequenced

Method used

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  • Methods of sequencing nucleic acids in mixtures and compositions related thereto
  • Methods of sequencing nucleic acids in mixtures and compositions related thereto
  • Methods of sequencing nucleic acids in mixtures and compositions related thereto

Examples

Experimental program
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Embodiment 1

[0316] Example 1: Sequencing mRNA using labeling reagents

[0317] Isolation of poly-AmRNA from cells and tissues using standard kits and removal of residues of genomic DNA is routine (DNA-Free™, Life Technology).

[0318] 1. Reverse transcribe cDNA from RNA (Moloney Murine Leukemia Virus Reverse Transcriptase), prime with a labeling reagent containing SMID; treat the heteroduplex with RNase H. Moloney murine leukemia virus reverse transcriptase may be replaced by other viral reverse transcriptases or equivalent enzymes from any other source capable of reverse transcription of RNA.

[0319] 2. Circularize labeled single-stranded cDNA (T4RNA, DNA ligase (CircLigase; Epicentre)); use Exonuclease I to remove remaining linear cDNA.

[0320] 3. Divide the circularized cDNA suspension into aliquots and subject them to rolling circle amplification (RCA) (phi29 DNA polymerase) [The population of cDNA to be amplified may vary with the choice of primers. ]

[0321] 4. Optionally debr...

Embodiment 2

[0323] Example 2: Non-polyadenylated RNA

[0324] Salzman, J. et al reported in PloSOne, 2012, vol7, issue2, e30733 that circular RNAs are major transcript isoforms of hundreds of human genes in various cell types. These RNAs are not polyadenylated. Such RNA products are suitable for sequencing by this technique using labeling reagents of low stoichiometry with random 3' end sequences to obtain copies of the RNA, which are then circularized and processed as described herein.

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Abstract

This disclosure relates to analyzing the end-to-end sequence and the relative distributions in heterogeneous mixtures of polynucleotides and methods and enabling reagents related thereto. In certain embodiments this method relates to the complete full length sequencing and quantitative profiling of mRNAs present in the transcriptomes of cells or tissues of, but not limited to, higher multicellular organisms that possess interrupted genes subject to complex post-transcriptional RNA processing.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Application No. 61 / 766,841, filed February 20, 2013, which is hereby incorporated by reference in its entirety. Background technique [0003] A single gene can often produce new proteins in different cells or stages of differentiation, including cells not normally encountered during the life cycle of an organism (e.g., cancer cells, cells in culture, cells in developing neuroanatomical abnormalities ). The different proteins arise from differential patterns of transcriptional activation and post-transcriptional RNA processing of messenger RNA (mRNA) that specify the protein in the expressing cell. [0004] The population of mRNA "transcripts" present in a cell is referred to herein as the "transcriptome". The current state-of-the-art technology for transcriptome sequencing is "RNA-Seq". See Nature Methods (2008) 5, 621-628. In this method, mRNA isolated from tissue o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/10
CPCC12Q1/6869C12N15/1065C12Q2521/313C12Q2525/173C12Q2525/191C12Q2525/301C12Q2531/125
Inventor M·C·埃默里克W·S·安格纽
Owner EMORY UNIVERSITY
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