Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1992-05-19
1996-06-11
Zitomer, Stephanie W.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
4351723, C12Q 168, C12N 1563
Patent
active
055254934
DESCRIPTION:
BRIEF SUMMARY
This invention relates to a cloning method and to a kit for performing same.
The ability to splice genes, gene fragments or other target DNA into vectors or other pieces of DNA using restriction enzymes (RE) and ligases has been an important aspect in the advance of molecular biology and biotechnology. However, the technology of recombination or gene splicing has several disadvantages. Firstly, there is the need for conveniently situated restriction sites and often sites have to be constructed which not only takes time but can lead, in the long term, to mismatched reading frames and for example non-expression of a gene of interest. Such sites are usually introduced by means of oligonucleotide linkers which have to be synthesised and purified and are then used in excess to ensure the addition of the required RE sites(s) on to the target DNA. Secondly, the in vitro ligation or splicing of DNA is rather inefficient and relatively cumbersome screening techniques are required to locate desired recombinants. Finally, the technology is time-consuming and is not well-suited to automation. Accordingly, there is a need for a simple and relatively rapid method of cloning which avoids the problems of conventional splicing and the use of conventional plasmids.
It should be noted that conventional plasmids for cloning normally take the form of double stranded cyclic plasmid structures containing a promoter region separated from a gene or other DNA sequence of interest for replication or expression by one or more RE sites which permit the DNA of interest to be excised subsequently; such sites are also used for insertion of the DNA of interest for replication or expression, via one or more (RE) sites in the linearised plasmid which permit the introduction of DNA of interest, which is provided with `sticky ends` corresponding to RE sites of the linearised plasmid. When DNA has been synthesised, for example by cDNA synthesis from mRNA, by mutagenesis or by chemical synthesis, it is in single stranded form which is then conventionally treated with a polymerase to synthesise the second strand, provided with the required `sticky ends`, inserted into the double stranded plasmid vector and ligated to join covalently the insert to the vector which is then used to transform a host microorganism, e.g. E. Coli.
In recent years, the polymerase chain reaction (PCR) has been used for the amplification of target DNA. While this produces increased amounts of the DNA, it is often required to produce larger quantities by cloning in a suitable vector using a host microorganism such as E. Coli. Furthermore, for production of the corresponding protein it is required to incorporate the DNA into an expression vector.
For the reasons given above, conventional techniques for splicing the target gene into plasmid vectors are time consuming and inefficient and not well suited to automation. In cases where PCR itself is effected by an automated technique, it would be desirable for incorporation into the vector also to be readily added on to the automated system.
The present invention has as an object a method which provides for the formation of recombinant DNA from PCR amplified DNA without the need for restriction enzymes or ligases or the provision of restriction sites.
Accordingly, the present invention provides, in one aspect thereof, a method of amplifying target DNA wherein said DNA is first amplified by PCR, the amplified DNA then being contacted with a single stranded linearised plasmid vector having terminal which are complementary to terminal regions of the PCR amplified DNA, whereby a cyclic product is formed comprising single stranded sequences from said target DNA and said vector and two double stranded regions from the overlapping terminal regions of the vector and the PCR amplified DNA; the cyclic product then being introduced into a host organism.
It is surprising that the cyclic product can be used to transform a host directly; the native enzyme system of the host organism is capable of chain extension to complete synthesis of the d
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Hornes Erik
Uhlen Mathias
Dynal A/S
Zitomer Stephanie W.
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