Genetic Engineering - using recombinant DNA technology to modify an organism’s DNA to achieve desirable traits.
Recombinant DNA technology is a technology of joining together of DNA molecules from different species by laboratory methods of genetic recombination. The result of these genetic manipulations is recombinant DNA (rDNA) molecule or vector.
Addition of foreign DNA in the form of recombinant DNA vectors that are generated by molecular cloning is the most common method of genetic engineering.
An organism that receives the recombinant DNA is called a genetically modified organism (GMO).
If the foreign DNA that is introduced comes from a different species, the host organism is called transgenic.
Today, geneticists often use the term “chimera” to describe genetically engineered organisms that contain genes from unrelated species.
The first chimeric organism was created in 1973 when Stanley Cohen and Herbert Boyer successfully developed a bacterial plasmid that could express an amphibian gene.
Recombinant DNA Technology Steps
To insert a mammalian gene into a prokaryotic cell, two basic requirements must be met.
- First, researchers must isolate the target mammalian gene from the genome as a whole.
- Second, the researchers must find a way to ensure that the prokaryotic cell can express the mammalian gene correctly.
Creating and Isolating the Target Gene
The eukaryotic chromosome and selected bacterial plasmids to be used as a cloning vector are treated with a restriction endonuclease.
When the eukaryotic DNA fragments are combined with the broken plasmids, some of the plasmids recombine with eukaryotic DNA.
The plasmids are then returned to the host bacteria by simply culturing both in solution so that some of the bacteria will take up the plasmids.
However, many of the plasmids will not contain recombinant DNA; of those that do, only a small portion will contain the target mammalian gene.
Therefore, the next step is to isolate bacterial colonies that contain the recombinant plasmids incorporating the target gene.
This step involves two stages of screening:
Stage 1: Identify the bacterial colonies that contain recombinant plasmids.
Only a portion of the bacteria will take up recombinant plasmids.
To identify those that do, researchers typically use plasmids carrying a particular genetic marker — that is, a trait that is easily identified.
Stage 2: Identify the bacteria containing the desired gene.
When the mammalian DNA is broken with an endonuclease, the result is likely to be hundreds or thousands of fragments. Of these, only a small fraction will contain the target gene.
As a result, another step is required to find those bacteria that contain a plasmid that includes the right gene.
Identifying these bacteria involves the use of a nucleic acid probe in a technique called nucleic acid hybridization.
If at least part of the nucleic acid sequence of the gene is known, this information can be used to construct a probe made of RNA or single-stranded DNA. The probe consists of a nucleic acid sequence complementary to the known gene sequence, along with a radioactive or fluorescent tag.
To employ the probe, DNA from each bacterial colony is first heated to separate its two strands and then mixed with a solution containing the nucleic acid probe.
The probe forms a base pair with its complementary sequence, making it possible for researchers to locate the tag to determine which bacterial colony contains the desired gene.
Once the colony has been identified, it can be cultured to produce the gene product.
Expressing Eukaryotic Genes in Prokaryote Vectors
First, the promoter sequence of a eukaryotic gene will not be recognized by the prokaryotic form of RNA polymerase.
To overcome this problem, researchers have developed a particular type of plasmid called an expression vector.
An expression vector is a plasmid that contains a prokaryotic promoter sequence just ahead of a restriction enzyme target site. Thus, when recombination occurs, the inserted DNA sequence will lie close to the bacterial promoter. The host cell then recognizes the promoter and transcribes the gene.
Second, a prokaryote does not contain the snRNA or spliceosomes necessary to remove introns from a eukaryotic pre-mRNA transcript.
This means that the mRNA transcript in a prokaryote will contain both coding and non-coding sequences, both of which will be translated by the cell.
The solution to this problem has been to develop artificial eukaryotic genes that do not contain introns.
Researchers first isolate finished mRNA from the cytoplasm of an eukaryotic cell.
The mRNA is then placed in a solution with an enzyme called reverse transcriptase, which creates a DNA strand complementary to the mRNA strand.
This DNA strand is then isolated and added to a solution containing DNA polymerase, which synthesizes another complementary DNA strand.
The result is a double-stranded molecule of DNA containing only the coding portions of the eukaryotic gene. This synthetic molecule is called copy DNA or cDNA.
Another solution to both of these problems is to use eukaryotic cells as cloning vectors.
Yeast cells are often used for this purpose, since they can be cultured easily. Some yeast cells also contain plasmids, so similar techniques can be used to insert recombinant DNA into the cloning vector.
Inserting DNA into Plant or Animal Vectors
In some cases, only plant or animal cells will contain all the enzymes necessary to correctly manufacture a desired protein. Such cells can be grown in cultures to serve as cloning vectors.
However, because these cells are more difficult to culture, it is harder to insert foreign DNA into them.
To get around this apparent barrier and place foreign genes into eukaryotic genomes, biologists have developed several methods.
- Ti (for tumour-inducing) plasmid
- brief electric current
- DNA particle gun
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