Genetic Engineering: Techniques and Applications
DNA Fragmentation
Fragmentation of DNA: Enzymes capable of breaking the DNA are called restriction endonucleases, restrictases, or restriction enzymes. They are able to recognize and cut at specific points in the sequence of nucleotides, which are called target sequences.
Use of a Vector
A vector is responsible for introducing a particular piece of DNA into other cells.
Plasmids
Bacteria possess a circular, double-stranded DNA molecule in which they carry most of their genetic information. But they also have small molecules of double-stranded, circular DNA, called plasmids, and there can be multiple copies in a single bacterium.
Union of a DNA Fragment to a Vector: Recombinant DNA
The technique involves cutting the passenger DNA and the vector with the same restriction enzyme.
Introduction of Foreign DNA
Once the vector with the foreign DNA is obtained, it must incorporate it into a living cell so that it has to incorporate it into its genetic heritage. The technique employed will vary according to the vector used and the type of receptor cell. Transformation in bacteria is achieved by altering the environmental conditions to increase the permeability of the membrane to the DNA. If the vector is a virus, it already has the mechanisms for the introduction of its own and alien DNA. When using the Ti plasmid for plant cells, the bacteria has its own mechanisms for introducing the plasmid. Transformation is the introduction of extra DNA into bacteria, naturally or provided by vectors. If introduced into eukaryotic cells, the term transfection is used. If the inoculation of foreign DNA takes place in the reproductive cells of animals or plants, it is called transgenic, and these organisms are called transgenic organisms.
Cloning of DNA
A fragment has been achieved throughout the genome of a species (e.g., human) to introduce the fragments into bacteria using plasmid vectors and obtain clones of bacteria, each containing a particular restriction fragment. Therefore, we have DNA clones.
Cloning of a Gene
The clone is located that contains the appropriate fragment. Then it turns to the fragment, and the small fragment that contains the gene is located. Obtaining a clone of a particular gene is of great importance, especially when it is to seek a particular protein of interest, such as insulin, from bacteria that contain and express the gene.
Expression of Incorporated Genes
The possibility of introducing a gene into bacteria may serve to clone the gene. This seems even more interesting than the possibility that the gene is expressed and gives rise to the corresponding protein product (after the processes of transcription and translation).
Eukaryotic Genes
- Eukaryotic genes have introns, and those of bacteria do not. Instead of entering the original fragment of human DNA into the bacterium, what has been done is the following, without the information of introns:
- Isolate the mRNA of insulin.
- Synthesize a complementary DNA through reverse transcriptase.
- This DNA copy of the mature mRNA lacks introns.
- The cDNA is inserted into a vector and transformed into bacteria (recombinant DNA).
- cDNA lacks the promoter and other sequences of interest. The cDNA obtained from mRNA lacks the promoter and terminator; thus, there is no gene transcription in bacteria. Even if the inserted DNA had a promoter and terminator, these regions would not serve, for these areas are different between bacteria and eukaryotes. The only solution was to cut the promoter sequences of bacteria and join them to the human cDNA.
Sequencing DNA
Namely, the order in which each of the nucleotides in DNA are.
Therapeutic Protein Production by Bacteria
Insulin, growth hormone, interferon, Factor VIII coagulation, and vaccines.
Gene Therapy
Gene therapy is to introduce the correct gene into cells of a living being that have a mutation that causes disease. Somatic cell therapy is being carried out in three different ways:
- Ex vivo therapy (outside the body): Cells with defective genes are extracted, and vectors will be introduced through appropriate normal gene copies. Transfected cells are then implanted into the living being.
- In situ therapy (in the same place): Vectors are introduced together with the corrective genes directly into the tissues where these genes are needed. This technique is being tried to treat cystic fibrosis, a genetic disease caused by a mutation of a single gene that affects the lungs, seriously hindering breathing.
- In vivo therapy: This is the hope of the future. The appropriate gene vectors are injected into the blood.
Genetic Engineering Applications in Medicine
Genetic screening, legal medicine, and forensic medicine in crimes with no witnesses and where victims die or cannot recognize the aggressor.
Applications of Genetic Engineering in Agriculture: Transgenic Plants
The main applications of genetic engineering in agriculture will focus on obtaining the plants called transgenic. The introduction of foreign genes in eukaryotic cells is more complex than in bacteria. Some of the main achievements are:
- Development of transgenic varieties of maize.
- Obtaining transgenic wheat varieties with improved nutrition and resistance to pests and herbicides.
- Obtaining a variety of tomato that matures slowly.
Applications of Genetic Engineering in Animal Production: Transgenic Animals
The main applications of genetic engineering to improve animal production have been conducted in fish.
- GM Trouts: Grow between 20 and 46% faster due to the gene of growth hormone in rainbow trout.
- Transgenic Salmon: Better withstand low temperatures by incorporating a gene from a species that lives in the Arctic.
Human Genome Project
Knowledge of the normal sequence of nucleotides will be a reference for gene therapy. Work has a way of knowing the order in which genes are and the distance between them (human gene map). Another way is to decipher the nucleotide sequence (sequencing) of each gene. It is 100% fully sequenced.