Compounds and methods for molecular biology
AssigneeInvitrogen Corporation
InventorsDevon Byrd, Alice Young, James Hartley
Life of this patent
Estimating the term…
Abstract
The present invention provides materials and methods for the utilization of the specific interaction of replication termination sequences with their binding proteins in molecular biology applications.
Claims98
An isolated nucleic acid molecule engineered to comprise all or a portion of at least two Ter sites, wherein the nucleic acid comprises an origin of replication and the Ter sites are arranged with respect to the origin of replication such that the sequence between the two Ter sites is not replicated.
A modified Ter-binding protein.
A support comprising at least one oligonucleotide that comprises all or a portion of a Ter site.
A support comprising all or a portion of a Ter-binding protein.
A method for directional cloning, comprising:
- providing a nucleic acid molecule comprising one or more Ter sites or portions thereof;
- providing a vector molecule comprising one or more Ter sites or portions thereof;
- inserting the nucleic acid molecule into the vector molecule;
- and
- selecting the vector molecule comprising the nucleic acid molecule in the desired orientation.
A method for attaching a nucleic acid to a solid support, comprising:
- attaching all or a portion of one or more Ter-binding proteins to a solid support;
- and
- contacting the Ter-binding protein with a first nucleic acid, said nucleic acid comprising a Ter site.
A method of improving the transfection efficiency of a nucleic acid molecule, comprising:
- providing all or a portion of one or more Ter site in the nucleic acid molecule;
- and
- contacting the nucleic acid molecule with all or a portion of one or more Ter-binding proteins.
A method for improving the stability of a linear nucleic acid molecule in vivo, comprising:
- providing a linear nucleic acid molecule, the nucleic acid molecule comprising all or a portion of one or more Ter sites;
- contacting the nucleic acid molecule with all or a portion of one or more Ter-binding proteins to form a stable nucleic acid-protein complex;
- and
- introducing the stable nucleic acid-protein complex into a host cell, wherein the complex is more stable than the nucleic acid transfected alone.
A method for detecting a biological molecule, comprising:
- contacting a biological molecule with a reagent, said reagent comprising a nucleic acid portion and a portion that is capable of forming a specific complex with the biological molecule to form a detection mixture;
- contacting the detection mixture with a nucleic acid binding protein comprising a detection molecule, wherein the nucleic acid binding protein specifically binds to the nucleic acid portion of the reagent;
- and
- determining the presence or absence of the detection molecule in the detection mixture, wherein presence of the detection molecule correlates to presence of the biological molecule and absence of the detection molecule correlates to absence of the biological molecule.
A composition comprising all or a portion of one or more Ter-binding proteins attached to a support.
A method for separating a nucleic acid containing all or a portion of one or more Ter sites from a mixture, comprising:
- contacting the nucleic acid with a composition comprising all or a portion of a one or more Ter-binding proteins, wherein the Ter-binding protein binds to the Ter site;
- and
- separating the bound nucleic acid from the mixture.
A kit comprising one or more molecules selected from the group consisting of a nucleic acid molecule engineered to comprise all or a portion of at least two Ter sites and a polypeptide comprising all or a portion of one or more Ter-binding proteins.
The kit according to 62, further comprising at least one recombination protein.
A method of juxtaposing a Ter site on a nucleic acid molecule with a second site on the nucleic acid molecule, comprising:
- providing a nucleic acid molecule having a Ter site;
- contacting the nucleic acid with a Ter-binding protein in functional association with an enzyme capable of translocating along the nucleic acid molecule;
- and
- conducting a reaction that causes the enzyme to translocate, thereby juxtaposing the Ter site and the second site.
A method of cloning, comprising;
- providing a linear vector comprising a portion of a Ter site on each end;
- ligating a nucleic acid of interest with the vector to form a ligation mixture, wherein vectors that do not ligate with a nucleic acid reform a functional Ter site;
- and
- introducing the ligation mixture into host cells, wherein host cells that receive a vector with a functional Ter site do not replicate the vector.
A method for synthesizing a double stranded nucleic acid molecule comprising all or a portion of one or more Ter sites, comprising:
- (a) mixing one or more nucleic acid templates with a polypeptide having polymerase activity and one or more primers comprising all or a portion of one or more Ter sites;
- (b) incubating said mixture under conditions sufficient to synthesize a first nucleic acid molecule which is complementary to all or a portion of said templates and which comprises said all or portion of one or more Ter sites;
- and
- (c) incubating said first nucleic acid molecule in the presence of one or more primers under conditions sufficient to synthesize a second nucleic acid molecule complementary to all or a portion to said first nucleic acid molecule, thereby producing a double stranded nucleic acid molecule comprising all or a portion of one or more Ter sites.
A method for synthesizing a double stranded nucleic acid molecule comprising:
- mixing one or more nucleic acid templates with a polypeptide having polymerase activity and one or more primers comprising all or a portion of at least a first Ter site;
- incubating said mixture under conditions sufficient to synthesize a first nucleic acid molecule which is complementary to all or a portion of said one or more templates and which comprises at least said all or portion of a first Ter site;
- and
- incubating said first nucleic acid molecule in the presence of one or more primers under conditions sufficient to synthesize a second nucleic acid molecule complementary to all or a portion to said first nucleic acid molecule, thereby producing a double stranded nucleic acid molecule comprising all or a portion of at least a first Ter site, wherein said all or portion of a first Ter site comprises at least one nucleotide sequence that has at least 80-99% homology to a nucleotide sequence selected from the group of sequences in Table 4 and a corresponding or complementary DNA or RNA sequence.
A method for adding one or more Ter sites or portions thereof to one or more nucleic acid molecules, said method comprising:
- (a) contacting one or more nucleic acid molecules with one or more integration sequences which comprise one or more Ter sites or portions thereof;
- and
- (b) incubating said mixture under conditions sufficient to incorporate said integration sequences into said nucleic acid molecules.
A method for producing one or more cDNA molecules or a population of cDNA molecules comprising
- (a) mixing an RNA template or population of RNA templates with a reverse transcriptase and one or more primers wherein said primers comprise one or more Ter sites or portions thereof;
- and
- (b) incubating said mixture under conditions sufficient to make a first DNA molecule complementary to all or a portion of said template, thereby forming a first DNA molecule comprising one or more Ter sites or portions thereof.
A method for synthesizing one or more nucleic acid molecules comprising all or a portion of one or more Ter sites, said method comprising:
- (a) obtaining one or more linear nucleic acid molecules;
- and
- (b) contacting said molecules with one or more adapters which comprise one or more Ter sites or portions thereof under conditions sufficient to add one or more of said adapters to one or more termini of said linear nucleic acid molecule.
A nucleic acid molecule comprising all or a portion of a Ter site flanked by recombination sites.
A method of cloning two DNA fragments into one vector in one reaction, wherein said vector comprises two markers for negative selection, said method comprising:
- replacing a first marker for negative selection with a first DNA fragment;
- in the same reaction mixture, replacing a second marker for negative selection with a second DNA fragment;
- and
- transforming host cells that are not resistant to either negative selection.
Dependencies are inferred from the claim text.