US 2018/0023089 A1 · Methods for Dynamic Vector Assembly of DNA Cloning Vector Plasmids

Abstract

A method for using cloning vector plasmids to produce DNA molecules, such as transgenes, in a single cloning step. The transgenes can be used for the purpose of gene expression or analysis of gene expression. The plasmid cloning vectors are engineered to minimize the amount of manipulation of DNA fragment components by the end user of the vectors and the methods for their use. Transgenes produced using the invention may be used in a single organism, or in a variety of organisms including bacteria, yeast, mice, and other eukaryotes with little or no further modification.

Claims35

Dependent claims

(canceled)

A method for making a modular cloning vector plasmid for the synthesis of a transgene or other DNA construct, the method comprising:

(a) providing a cloning vector plasmid comprising a backbone, the backbone comprising first and second docking points, each docking point being fixed within the backbone and comprising at least one endonuclease site of greater than six nucleotides, wherein the cloning vector plasmid further comprises a unique homing endonuclease site in a forward orientation located upstream from the 5′ end of the first docking point and a unique homing endonuclease site in a reverse orientation located downstream from the 3′ end of the second docking point;
(b) cleaving the first docking point with a first endonuclease enzyme corresponding to the at least one endonuclease site of greater than six nucleotides of the first docking point, leaving the cleaved first docking point with a 3′ end;
(c) cleaving the second docking point with a second nuclease enzyme corresponding to the at least one endonuclease site of greater than six nucleotides of the second docking point, leaving the cleaved second docking point with a 5′ end;
(d) providing at least a first and a second insert, each insert comprising a 5′ end, a nucleotide sequence of interest and a 3′ end, wherein the 5′ end of the first insert is compatible with the 3′ end of the cleaved first docking point, the 3′ end of the second insert is compatible with the 5′ end of the cleaved second docking point, the 3′ end of the first insert being compatible with the 5′ end of the second insert to form a third endonuclease site of greater than six nucleotides for a third endonuclease enzyme;
and
(e) placing the inserts and the cleaved cloning vector plasmid into an appropriate reaction mixture to cause simultaneous ligation and self-orientation of the first and second inserts between the first and second docking points within the backbone, thereby re-forming the first and second docking points, thereby forming the modular cloning vector plasmid.

3 more elements

A method for synthesizing a transgene or other DNA construct, comprising:

(a) providing a primary cloning vector plasmid comprising a backbone, the backbone comprising at least a first docking point and a second docking point, each docking point being fixed within the backbone and comprising at least one endonuclease site of at least six nucleotides for a non-variable rare restriction enzyme, wherein the cloning vector plasmid further comprises a unique homing endonuclease site in a forward orientation located upstream from the 5′ end of the first docking point and a unique homing endonuclease site in a reverse orientation located downstream from the 3′ end of the second docking point;
(b) cleaving the first docking point with a first non-variable rare restriction enzyme corresponding to one of the endonuclease sites of greater than six nucleotides of the first docking point, leaving the cleaved backbone with a 3′ end;
(c) cleaving the second docking point with a second non-variable rare restriction enzyme corresponding to one of the endonuclease sites of greater than six nucleotides of the second docking point, leaving the cleaved backbone with a 5′ end;
(d) providing a Promoter insert into which a Promoter sequence of interest, a 5′ end that is compatible with the 3′ end of the first docking point, and a 3′ end;
(e) providing an Expression insert comprising an Expression sequence of interest, a 5′ end that is compatible with the 3′ end of the Promoter insert to form a endonuclease site of greater than six nucleotides for a third non-variable rare restriction enzyme, and a 3′ end;
(f) providing a Regulatory insert comprising a Regulatory sequence of interest, a 5′ end that is compatible with the 3′ end of the Expression insert to form a endonuclease site of greater than six nucleotides for a fourth non-variable rare restriction enzyme, and a 3′ end that is compatible with the 5′ end of the cleaved second docking point;
and
(g) placing the Promoter, Expression and Regulatory inserts and the cleaved cloning vector plasmid into an appropriate reaction mixture to cause simultaneous ligation, self-orientation and sequential placement of the Promoter, Expression and Regulatory inserts between the first and second docking points, reforming the first and second docking points, and forming a modular primary cloning vector plasmid.

5 more elements

A method for simultaneously synthesizing an array of transgenes or other DNA constructs, comprising:

(a) providing at least one primary cloning vector plasmid comprising a backbone into which inserts having a 5′ end, a nucleotide sequence of interest and a 3′ end can be inserted, the backbone operable to accept a sequential arrangement of Promoter, Expression, and Regulatory inserts and comprising at least a first and a second docking point, each docking point being fixed within the backbone and comprising at least one endonuclease site of greater than six nucleotides for a non-variable rare restriction enzyme, wherein the cloning vector plasmid further comprises a unique homing endonuclease site in a forward orientation located upstream from the 5′ end of the first docking point and a unique homing endonuclease site in a reverse orientation located downstream from the 3′ end of the second docking point;
(b) cleaving the first docking point with a first non-variable rare restriction enzyme corresponding to one of the endonuclease sites of greater than six nucleotides of the first docking point;
(c) cleaving the second docking point with a second non-variable rare restriction enzyme corresponding to one of the endonuclease sites of greater than six nucleotides of the second docking point;
(d) providing at least one Promoter insert into which a Promoter nucleotide sequence has been inserted, the 5′ end of the at least one Promoter insert compatible with the 3′ end of the cleaved first docking point;
(e) providing at least one Expression insert into which an Expression nucleotide sequence has been inserted, the 5′ end of the at least one Expression insert being compatible with the 3′ end of the at least one Promoter insert to form an endonuclease site of greater than six nucleotides for a third non-variable rare restriction enzyme;
(f) providing at least one Regulatory insert into which a Regulatory nucleotide sequence has been inserted, the 5′ end of the at least one Regulatory insert being compatible with the 3′ end of the at least one Expression insert to form endonuclease site of greater than six nucleotides for a fourth non-variable rare restriction enzyme, the 3′ end of the at least one Regulatory insert being compatible with the 5′ end of the of the cleaved second docking point;
and
(g) placing at least two different types of at least one of the Promoter, Expression and Regulatory inserts, at least one of each of the remaining inserts, and the cleaved cloning vector plasmid into an appropriate reaction mixture to cause simultaneous ligation, self-orientation and sequential placement of one each of the Promoter, Expression and Regulatory inserts between the first and second docking points within the backbone, thereby creating an array of plasmids having different combinations of Promoter, Expression and Regulatory inserts within their backbone.

5 more elements

A method of preparing one or more transgenes comprising:

(a) providing a cloning vector plasmid comprising at least a first and a second docking point, wherein said first and said second docking points each comprise a nucleotide sequence that is recognized by at least a first and a second rare restriction enzyme, respectively, and wherein said first and said second rare restriction enzyme are different from each other, and wherein the cloning vector plasmid further comprises a unique homing endonuclease site in a forward orientation located upstream from the 5′ end of the first docking point and a unique homing endonuclease site in a reverse orientation located downstream from the 3′ end of the second docking point;
(b) digesting said cloning vector plasmid using said first and said second rare restriction enzyme to obtain a digested vector having a 5′ end and a 3′ end;
(c) providing at least a first and a second polynucleotide insert wherein each of said first and second polynucleotide inserts comprises a 5′ insert end, a nucleotide sequence of interest and a 3′ insert end;
(d) placing said first polynucleotide insert and said second polynucleotide insert and said digested vector together into an appropriate reaction mixture to allow simultaneous annealing and ligating of said inserts and said digested vector, thereby obtaining an insert-bearing vector,
wherein said first polynucleotide insert and said second polynucleotide insert and said digested vector anneal and ligate with each other in a predetermined order and orientation such that said first docking point of said cloning vector is re-formed between said 5′ end of said first polynucleotide insert and said 3′ end of said digested vector, and said second docking point of said cloning vector is re-formed between said 3′ insert end of said second insert and said 5′ end of said digested vector,
wherein at least one additional docking point is formed between the re-formed first and re-formed second docking points, and
wherein each additional docking point comprises a nucleotide sequence that is recognized by an additional rare restriction enzyme that is different from said first rare and said second rare restriction enzymes.

4 more elements

Dependencies are inferred from the claim text.

Facts

Application

14/825,195Filed 13 Aug 2015

Priority

18 May 2004

Published

25 Jan 2018

Term ends

Not estimatedThe source records don’t support an estimate.

Classification6 codes

C12N 15/64
Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression: general methods for preparing…
C07H 21/04
Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of…: with…
C12N 15/66
Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression: general methods for inserting a…
C12N 15/85
Vectors or expression systems specially adapted for eukaryotic hosts: for animal cells

Family

Application60/572,011Us provisional application · filed 18 May 2004

Application11/569,335Continuation · filed 22 Feb 2007

Application14/532,088Continuation · filed 4 Nov 2014

Application14/825,195Continuation

US 10,036,026 B2Related patent

US 2009/0170727 A1Published application

US 2015/0057192 A1Published application

US 9,115,361 B2Related patent

US 2018/0023089 A1This patent

From the US filing history. Foreign family members aren’t listed.

Abstract

Claims35

Dependent claims

(canceled)

A method for making a modular cloning vector plasmid for the synthesis of a transgene or other DNA construct, the method comprising:

(a) providing a cloning vector plasmid comprising a backbone, the backbone comprising first and second docking points, each docking point being fixed within the backbone and comprising at least one endonuclease site of greater than six nucleotides, wherein the cloning vector plasmid further comprises a unique homing endonuclease site in a forward orientation located upstream from the 5′ end of the first docking point and a unique homing endonuclease site in a reverse orientation located downstream from the 3′ end of the second docking point;
(b) cleaving the first docking point with a first endonuclease enzyme corresponding to the at least one endonuclease site of greater than six nucleotides of the first docking point, leaving the cleaved first docking point with a 3′ end;
(c) cleaving the second docking point with a second nuclease enzyme corresponding to the at least one endonuclease site of greater than six nucleotides of the second docking point, leaving the cleaved second docking point with a 5′ end;
(d) providing at least a first and a second insert, each insert comprising a 5′ end, a nucleotide sequence of interest and a 3′ end, wherein the 5′ end of the first insert is compatible with the 3′ end of the cleaved first docking point, the 3′ end of the second insert is compatible with the 5′ end of the cleaved second docking point, the 3′ end of the first insert being compatible with the 5′ end of the second insert to form a third endonuclease site of greater than six nucleotides for a third endonuclease enzyme;
and
(e) placing the inserts and the cleaved cloning vector plasmid into an appropriate reaction mixture to cause simultaneous ligation and self-orientation of the first and second inserts between the first and second docking points within the backbone, thereby re-forming the first and second docking points, thereby forming the modular cloning vector plasmid.

3 more elements

A method for synthesizing a transgene or other DNA construct, comprising:

(a) providing a primary cloning vector plasmid comprising a backbone, the backbone comprising at least a first docking point and a second docking point, each docking point being fixed within the backbone and comprising at least one endonuclease site of at least six nucleotides for a non-variable rare restriction enzyme, wherein the cloning vector plasmid further comprises a unique homing endonuclease site in a forward orientation located upstream from the 5′ end of the first docking point and a unique homing endonuclease site in a reverse orientation located downstream from the 3′ end of the second docking point;
(b) cleaving the first docking point with a first non-variable rare restriction enzyme corresponding to one of the endonuclease sites of greater than six nucleotides of the first docking point, leaving the cleaved backbone with a 3′ end;
(c) cleaving the second docking point with a second non-variable rare restriction enzyme corresponding to one of the endonuclease sites of greater than six nucleotides of the second docking point, leaving the cleaved backbone with a 5′ end;
(d) providing a Promoter insert into which a Promoter sequence of interest, a 5′ end that is compatible with the 3′ end of the first docking point, and a 3′ end;
(e) providing an Expression insert comprising an Expression sequence of interest, a 5′ end that is compatible with the 3′ end of the Promoter insert to form a endonuclease site of greater than six nucleotides for a third non-variable rare restriction enzyme, and a 3′ end;
(f) providing a Regulatory insert comprising a Regulatory sequence of interest, a 5′ end that is compatible with the 3′ end of the Expression insert to form a endonuclease site of greater than six nucleotides for a fourth non-variable rare restriction enzyme, and a 3′ end that is compatible with the 5′ end of the cleaved second docking point;
and
(g) placing the Promoter, Expression and Regulatory inserts and the cleaved cloning vector plasmid into an appropriate reaction mixture to cause simultaneous ligation, self-orientation and sequential placement of the Promoter, Expression and Regulatory inserts between the first and second docking points, reforming the first and second docking points, and forming a modular primary cloning vector plasmid.

5 more elements

A method for simultaneously synthesizing an array of transgenes or other DNA constructs, comprising:

(a) providing at least one primary cloning vector plasmid comprising a backbone into which inserts having a 5′ end, a nucleotide sequence of interest and a 3′ end can be inserted, the backbone operable to accept a sequential arrangement of Promoter, Expression, and Regulatory inserts and comprising at least a first and a second docking point, each docking point being fixed within the backbone and comprising at least one endonuclease site of greater than six nucleotides for a non-variable rare restriction enzyme, wherein the cloning vector plasmid further comprises a unique homing endonuclease site in a forward orientation located upstream from the 5′ end of the first docking point and a unique homing endonuclease site in a reverse orientation located downstream from the 3′ end of the second docking point;
(b) cleaving the first docking point with a first non-variable rare restriction enzyme corresponding to one of the endonuclease sites of greater than six nucleotides of the first docking point;
(c) cleaving the second docking point with a second non-variable rare restriction enzyme corresponding to one of the endonuclease sites of greater than six nucleotides of the second docking point;
(d) providing at least one Promoter insert into which a Promoter nucleotide sequence has been inserted, the 5′ end of the at least one Promoter insert compatible with the 3′ end of the cleaved first docking point;
(e) providing at least one Expression insert into which an Expression nucleotide sequence has been inserted, the 5′ end of the at least one Expression insert being compatible with the 3′ end of the at least one Promoter insert to form an endonuclease site of greater than six nucleotides for a third non-variable rare restriction enzyme;
(f) providing at least one Regulatory insert into which a Regulatory nucleotide sequence has been inserted, the 5′ end of the at least one Regulatory insert being compatible with the 3′ end of the at least one Expression insert to form endonuclease site of greater than six nucleotides for a fourth non-variable rare restriction enzyme, the 3′ end of the at least one Regulatory insert being compatible with the 5′ end of the of the cleaved second docking point;
and
(g) placing at least two different types of at least one of the Promoter, Expression and Regulatory inserts, at least one of each of the remaining inserts, and the cleaved cloning vector plasmid into an appropriate reaction mixture to cause simultaneous ligation, self-orientation and sequential placement of one each of the Promoter, Expression and Regulatory inserts between the first and second docking points within the backbone, thereby creating an array of plasmids having different combinations of Promoter, Expression and Regulatory inserts within their backbone.

5 more elements

A method of preparing one or more transgenes comprising:

(a) providing a cloning vector plasmid comprising at least a first and a second docking point, wherein said first and said second docking points each comprise a nucleotide sequence that is recognized by at least a first and a second rare restriction enzyme, respectively, and wherein said first and said second rare restriction enzyme are different from each other, and wherein the cloning vector plasmid further comprises a unique homing endonuclease site in a forward orientation located upstream from the 5′ end of the first docking point and a unique homing endonuclease site in a reverse orientation located downstream from the 3′ end of the second docking point;
(b) digesting said cloning vector plasmid using said first and said second rare restriction enzyme to obtain a digested vector having a 5′ end and a 3′ end;
(c) providing at least a first and a second polynucleotide insert wherein each of said first and second polynucleotide inserts comprises a 5′ insert end, a nucleotide sequence of interest and a 3′ insert end;
(d) placing said first polynucleotide insert and said second polynucleotide insert and said digested vector together into an appropriate reaction mixture to allow simultaneous annealing and ligating of said inserts and said digested vector, thereby obtaining an insert-bearing vector,
wherein said first polynucleotide insert and said second polynucleotide insert and said digested vector anneal and ligate with each other in a predetermined order and orientation such that said first docking point of said cloning vector is re-formed between said 5′ end of said first polynucleotide insert and said 3′ end of said digested vector, and said second docking point of said cloning vector is re-formed between said 3′ insert end of said second insert and said 5′ end of said digested vector,
wherein at least one additional docking point is formed between the re-formed first and re-formed second docking points, and
wherein each additional docking point comprises a nucleotide sequence that is recognized by an additional rare restriction enzyme that is different from said first rare and said second rare restriction enzymes.

4 more elements

Dependencies are inferred from the claim text.

Methods for Dynamic Vector Assembly of DNA Cloning Vector Plasmids

Life of this patent

Abstract

Claims35

Methods for Dynamic Vector Assembly of DNA Cloning Vector Plasmids

Life of this patent

Abstract

Claims35