Genetics

How Recombinant Dna is Created



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Recombinant DNA is the result of combining DNA sequences that normally do not occur together. The first report of recombinant DNA technology was a 1974 paper by Cohen and Boyer. The 1978 Nobel Prize in Medicine went to the group (Arber, Nathans, and Smith) that discovered restriction nucleases, enzymes essential to recombinant technology. In the years since, the technology has become an integral part of genetic research.

The most common recombinant DNA is the insertion of genes into bacterial plasmids, circular DNA that contains all necessary components for the production of the recombinant protein. The genes may be from one organism or several. The final plasmid is the combination of the vector (original, clipped plasmid) and the inserted gene(s).

The key to recombinant DNA is the presence of short sequences in the DNA that are recognized by restriction endonucleases, enzymes that clip the double-stranded DNA. Some enzymes result in a blunt end, others leave an overhang that can join like a puzzle piece to another strand. This is the limitation to the technology, though there are many endonucleases to choose from, the presence of the particular recognition sequence in the gene of interest, as well as the plasmid chosen for use, is necessary. However, work-arounds have been found for labs to insert recognition sequences onto the ends of the gene of interest.

The first step in creating recombinant DNA is to identify and isolate the gene of interest from the genome. There are immense databases of sequence data from the human genome project, along with a number of other organisms commonly studied. Scientists design PCR primers that recognize sequences flanking the gene of interest that will result in the desired size and include the entire gene and its known regulatory sequences. After PCR (polymerase chain reaction, there is an accumulation of the gene as double-stranded DNA. Another important part of the primer design is the inclusion of endonuclease recognition sites. If there are none present in convenient parts of the native gene sequence, PCR primers can include it and insert it onto the ends of the PCR products, effectively attaching the recognition sites onto the ends of the gene.

The PCR products, essentially the gene of interest, are digested with the endonuclease(s). The use of two different endonucleases will guarantee the alignment of the gene in the plasmid by creating two different ends. The plasmid that the gene will be inserted into is also digested with the same enzymes. A particular plasmid is chosen to carry the gene of interest for several reasons. Scientists take into account the size; intended use gene expression, cloning, selection; reporter genes present that can be used for the selection of bacterial colonies; and the restriction sites available.

If the enzyme leaves a blunt end, then additional steps are carried out to create sticky ends for ligation into the plasmid. Otherwise, the cut plasmid and gene are ligated, combined, in a reaction with the enzyme ligase. The mixture is then put into bacterial cells by various methods and the cells plated.

On the agar plate growing the non-infectious bacterial cells is usually an antibiotic or reporter substance that the plasmid interacts with to sort out the plasmids containing the gene of interest and those not containing it. Positive colonies are then isolated and the experiments proceed depending on the aim. Sometimes it means isolation of the DNA for placement in a vector for in vitro experiments. Sometimes it is large scale culture of the bacteria for the production of the recombinant protein.

Recombinant DNA is used to determine the effects of mutations on gene expression, to determine the effect of the presence of a gene in disease, and to produce large quantities of proteins needed in medical therapy. It is a tedious process for scientists, often taking a week for the development of the final plasmid, which doesn't always work the first time.

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