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US 2002/0160377 A1Published application
Methods for producing nucleic acids lacking 3'-untranslated regions and optimizing cellular RNA -protein fusion formation
OwnerPhilip W. Hammond, Dasa Lipovsekinventors
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Abstract
Described herein are methods for removing the 3′-untranslated regions from cDNA or mRNA molecules, as well as methods for the use of such products for RNA-protein fusion formation.
Claims16
A library of nucleic acid molecules, each molecule comprising an open reading frame and lacking the 3′-untranslated region normally associated with said open reading frame.
A library of nucleic acid molecules produced by the steps of:
- (a) providing a library of DNA molecules, each having an open reading frame and a 3′-untranslated region, each of said DNA molecules terminating at its 5′ end in an overhang and at its 3′ end in a blunt end;
- and
- (b) treating said library of DNA molecules first with a 3′→5′ exonuclease and then with a single-stranded nuclease under conditions that allow removal of the 3′-untranslated regions of said DNA molecules.
A library of nucleic acid molecules produced by the steps of:
- (a) translating a library of mRNA molecules in vitro in a translation reaction mixture lacking functional translation release factor activity, resulting in pausing of the translation reaction mixture ribosomes at the stop codons of said mRNA molecules;
- (b) adding, to said translation reaction mixture of step (a), reverse transcriptase and oligonucleotide primers which are complementary to the 3′-untranslated regions of said mRNA molecules at a site proximal to said stop codons, under conditions which allow the synthesis of strands of DNA that are complementary to said 3′-untranslated regions and terminate at sites proximal to said stop codons;
- and
- (c) removing the RNA portions of the RNA-DNA duplexes formed in step (b), thereby removing the 3′-untranslated regions of said mRNA molecules.
1 more element
A library of nucleic acid molecules produced by the steps of:
- (a) providing a population of mRNA molecules;
- (b) synthesizing strands of DNA, each of which is complementary to one of said mRNA molecules, using a random primer mixture, said random primer mixture comprising primers, each having
- (i) a 3′ region comprising a stop codon flanked by a random oligonucleotide located 3′, 5′, or both to said stop codon;
- and
- (ii) a 5′ region comprising a Type IIS restriction site;
- (c) ligating to the 3′ ends of the DNA products of step (b) an oligonucleotide tail;
- (d) amplifying the products of step (c) using
- (i) a first primer which is complementary to said Type IIS restriction site-containing sequence;
- and
- (ii) a second primer which is complementary to said oligonucleotide tail;
- and
- (e) treating the products of step (d) with a Type IIS restriction enzyme that recognizes said Type IIS restriction site to cleave said products, thereby removing the 3′-untranslated regions and stop codons.
9 more elements
A library of nucleic acid molecules produced by the steps of:
- (a) providing a population of mRNA molecules;
- (b) synthesizing strands of DNA, each of which is complementary to one of said mRNA molecules, using a random primer mixture, said random primer mixture comprising primers, each having (i) a 5′ region which lacks a stop codon in at least one reading frame and (ii) a random 3′ region;
- and
- (c) synthesizing strands of DNA complementary to said DNA strands of step (b), using a second random primer mixture.
1 more element
Dependencies are inferred from the claim text.