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.

Description

BACKGROUND OF THE INVENTION[0001]The present invention relates in general to the field of cloning vector plasmids, and in particular to methods for rapidly assembling DNA constructs or transgenes with cloning vector plasmids.[0002]The foundation of molecular biology is recombinant DNA technology, which can here be summarized as the modification and propagation of nucleic acids for the purpose of studying the structure and function of the nucleic acids and their protein products.[0003]Individual genes, gene regulatory regions, subsets of genes, and indeed entire chromosomes in which they are contained, are all comprised of double-stranded anti-parallel sequences of the nucleotides adenine, thymine, guanine and cytosine, identified conventionally by the initials A, T, G, and C, respectively. These DNA sequences, as well as cDNA sequences, which are double stranded DNA copies derived from mRNA (messenger RNA) molecules, can be cleaved into distinct fragments, isolated, and inserted int...

AI Technical Summary

Benefits of technology

[0030]Accordingly, the present invention provides a method of rapidly assembling DNA constructs or transgenes by using cloning vector plasmids. The invention also provides a method that incorporates multiple DNA fragments, also known as both “inserts” or “modules”, such as one each of a Promoter, Expression, and 3′ Regulatory nucleotide sequence, into a cloning vector plasmid in a single step, rather than having to introduce each insert in a sequential manner. Such a method is called “Dynamic Vector Assembly” herein.

[0037]In yet another embodiment, the invention provides a method for simultaneously synthesizing an array of transgenes or other complicated DNA constructs, comprising the steps of: 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 restriction site for a non-variable rare restriction enzyme; cleaving the first docking point with a first non-variable rare restriction enzyme corresponding to one of the restriction sites of the first docking point; cleaving the second docking point with a second non-variable rare restriction enzyme corresponding to one of the restriction sites of the second docking point; 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 to the 3′ end of the first docking point which was cleaved in step ‘b’; 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 to the 3′ end of the at least one Promoter insert to form a restriction site for a third non-variable rare restriction enzyme; 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 to the 3′ end of the at least one Expression insert to form a restriction site for a fourth non-variable rare restriction enzyme, the 3′ end of the at least one Regulatory insert compatible to the 5′ end of the of the second docking point which was cleaved in step ‘c’; and thereafter 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.

Problems solved by technology

Any plasmid acquired must express a gene or genes that contribute to the survival of the host or else it will be destroyed or discarded by the organism, since the maintenance of unnecessary plasmids would be a wasteful use of resources.

When planning the construction of a transgene or other recombinant DNA molecule, this is a vital issue, since such a project frequently requires the assembly of several pieces of DNA of varying sizes.

The larger these pieces are, the more likely that the sites one wishes to use occur in several pieces of the DNA components, making manipulation difficult at best.

If a promoter sequence is 3000 bp and a gene of interest of 1500 bp are to be assembled into a cloning vector of 3000 bp, it is statistically very likely that many sites of 6 or less nucleotides will not be useful, since any usable sites must occur in only two of the pieces.

In addition, most cloning projects will need to have additional DNA elements added, thereby increasing the complexity of the growing molecule and the likelihood of inopportune repetition of any particular restriction site.

Since any restriction enzyme will cut at all of its sites in a molecule, if an endonuclease enzyme restriction site reoccurs, all the inopportune sites will be cut along with the desired sites, disrupting the integrity of the molecule.

Since most DNA constructs are designed for a single purpose, little thought is given to any future modifications that might need to be made, further increasing the difficulty for future experimental changes.

1. There is a wide variety of endonuclease enzymes available that will generate an array of termini, however most of these are not compatible with each other. Many endonuclease enzymes, such as EcoR1, generate DNA fragments with protruding 5′ cohesive termini or “tails”; others (e.g., Pst1) generate fragments with 3′ protruding tails, whereas still others (e.g., Ball) cleave at the axis of symmetry to produce blunt-ended fragments. Some of these will be compatible with the termini formed by cleavage with other endonuclease enzymes, but the majority of useful ones will not. The termini that can be generated with each DNA fragment isolation must be carefully considered in designing a DNA construct.

2. DNA fragments needed for assembly of a DNA construct or transgene must first be isolated from their source genomes, placed into plasmid cloning vectors, and amplified to obtain useful quantities. The step can be performed using any number of commercially-available or individually altered cloning vectors. Each of the different commercially available cloning vector plasmids were, for the most part, developed independently, and thus contain different sequences and endonuclease sites for the DNA fragments of genes or genetic elements of interest. Genes must therefore be individually tailored to adapt to each of these vectors as needed for any given set of experiments. The same DNA fragments frequently will need to be altered further for subsequent experiments or cloning into other combinations for new DNA constructs or transgenes. Since each DNA construct or transgene is custom made for a particular application with no thought or knowledge of how it will be used next, it frequently must be “retrofitted” for subsequent applications.

3. In addition, the DNA sequence of any given gene or genetic element varies and can contain internal endonuclease sites that make it incompatible with currently available vectors, thereby complicating manipulation. This is especially true when assembling several DNA fragments into a single DNA construct or transgene.

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