Methods for detecting modification resistant nucleic acids

Patent metadata

Field	Value
Publication number	US-9677068-B2
Application number	US-201615017214-A
Country	US
Kind code	B2
Filing date	Feb 5, 2016
Priority date	Nov 3, 2008
Publication date	Jun 13, 2017
Grant date	Jun 13, 2017

Abstract

Methods are provided for, inter alia, detecting nucleic acid molecules resistant to degradation, such as a plurality of RNA molecules bound to a ribosome, using various technologies including deep sequencing.

First claim

What is claimed is:

1. A method for determining a ribosome footprint density, comprising:
(a) isolating a plurality of monosomes from a plurality of polysomes, wherein each of said plurality of polysomes comprises a ribosome bound to a portion of a translatable RNA molecule;
(b) sequencing each of said portions; and
(c) determining a ribosome footprint density for each translatable RNA molecule from said sequencing, thereby determining a ribosome footprint density.

2. The method of claim 1 , wherein the determining of the ribosome footprint density comprises aligning each of said portions.

3. The method of claim 1 , further comprising quantifying each of said portions.

4. The method of claim 1 , wherein said isolating comprises contacting said plurality of polysomes with a degradant.

5. The method of claim 1 , further comprising quantifying a relative amount of two or more of said portions.

6. The method of claim 2 , further comprising determining a location of high ribosome footprint density within said translatable RNA molecule.

7. The method of claim 1 , wherein said sequencing is determined by using a sequence by synthesis technique.

8. The method of claim 1 , wherein said determining of said protein synthesis rate comprises determining expression of a plurality of protein coding genes expressed from said translatable RNA molecules.

9. The method of claim 8 , wherein said protein coding genes are located on two or more different chromosomes.

10. The method of claim 4 , further comprising removing ribosomal RNA prior to step (a).

11. The method of claim 1 , further comprising amplifying the translatable RNA portion prior to sequencing.

12. The method of claim 1 , further comprising determining a protein synthesis rate from said ribosomal footprint density.

Assignees

• Univ California

Inventors

• Weissman Jonathan
• Ingolia Nicholas
• Ghaemmaghami Sina
• Newman John

Classifications

• C12Q1/6809

  Methods for determination or identification of nucleic acids involving differential detection · CPC title

• C12N15/1041 Primary

  Ribosome/Polysome display, e.g. SPERT, ARM · CPC title

• C12N15/1093

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

• C40B40/08

  Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries · CPC title

• C40B50/06

  Biochemical methods, e.g. using enzymes or whole viable microorganisms · CPC title