Gene mutations are changes in the base sequence of DNA molecules. There are five types which include:
Insertion where bases are added, deletion where bases are lost. This type of mutation causes a frameshift of bases, triplets code change and a normal polypeptide chain cannot be produced. Duplication is when one or more bases are repeated and therefore produce a frame shift. Inversion mutation includes a group of bases that is separated and then re-join in the reverse order, which overall affects the amino acid being produced.
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Translocation mutation includes a group of bases that become separated from the DNA sequence on the chromosome and are inserted into the DNA sequence on another chromosome. This can lead to significant effects on the phenotype.
Gene mutations occur randomly during DNA replication, however, various factors in the environment such as mutagens can increase the mutation rate. Chemical mutagens include alkylating agents that transfer CH3 (methyl) and CH2CH3 (ethyl) groups to DNA molecules, changing their activity. Other chemical mutagens include asbestos and tar in tobacco.
Ionizing radiation such as alpha, beta, gamma, UV and X-rays can also affect DNA structure. Ionizing radiation damages DNA by removing electrons from the atoms of its molecules.
Overall mutations can have a neutral effect where the mutation causes no change to the organism e.g. the mutation occurs in a non-coding region of DNA or is a silent mutation. However, mutations in some cases can become beneficial for organisms. E.g. humans developed trichromatic vision through a mutation.
Genetically modified organisms (GMOs) are organisms that have had their DNA altered through recombinant DNA technology. Recombinant DNA technology involves the transfer of fragments of DNA from one organism to another. Transgenic organisms can successfully express genes from any organism as the genetic code and the mechanism of production are universal.
DNA fragments are created by: using restriction endonucleases to cut at recognition sites near the desired gene. The mRNA of the desired gene is converted into cDNA, using reverse transcriptase. Double-stranded DNA is then synthesized using DNA polymerase. A gene machine is used to synthesize the gene. The gene sequence is determined by the primary protein structure. The isolated gene is then modified by the addition of a promotor and a terminator region. A vector is used to transfer the isolated gene into a host cell which is mainly a plasmid. The plasmid and gene are cut with the same restriction enzyme to create complementary sticky ends. This means that the inserted DNA and vector can be joined. The fragments are incubated with the plasmids. If a plasmid takes up the insert, base pairing takes place between the complementary ends which are then sealed with the use of the DNA ligase which forms phosphodiester bonds.
A recombinant DNA molecule is created in the formation of transgenic microorganisms, electroporation is used to stimulate bacterial cells to take up plasmids. Electroporation facilitates the process by increasing permeability of bacterial membranes thus increasing the chance of success. This is achieved by the use of calcium salts and rapid temperature changes from 0 to 40 oc.
Gene markers are used to check whether the DNA has been taken up by the bacteria. There are different types of gene markers, one type is antibiotic-resistant genes and the other type is fluorescent markers and enzyme markers. These genes are inserted into the plasmid to distinguish where the desired DNA has entered the plasmid as the gene markers become inactivated. Another form of gene cloning is a polymerase chain reaction known as PCR, which is used to amplify DNA by making millions of copies of a given DNA sample.
PCR occurs as follows: A reaction mixture is set up by mixing the DNA sample, primers, free nucleotides, and DNA polymerase which is the enzyme involved in creating new DNA strands. The mixture is then heated to 95oc to break the hydrogen bonds and to separate the double strands. The mixture is then cooled to 55oc so the primers bind to the start of each template strand preventing the templates from re-joining and allowing DNA polymerase to bind to the new strand.
After that, the temperature is increased to 70oc, as this is the temperature DNA polymerase works. The DNA polymerase creates a copy of the sample by complementary base pairing using the free nucleotides. This cycle is repeated about 25-30 times and as a result over 50 million copies of the template DNA is formed.
This method of PCR cloning is called in vitro cloning which is fast, automated, and reliable once conditions are established. This does not require living cells and can have problems such as contamination and errors.
In-vivo gene cloning can be done using recombinant plasmids in bacteria. This is accurate and useful as the gene is placed in cells where it can be expressed. The disadvantage of recombinant DNA is that it is a time-consuming process and requires monitoring of cell growth.
Recombinant DNA technology has enabled the diagnosis and treatment of many genetic disorders and to enable this, it is necessary to locate the specific gene that is causing the disorder. This is done by DNA probes and DNA hybridization.
DNA probes is a short, single-stranded DNA molecule that has a specific base sequence that is complementary to a specific base sequence of a specific allele. The two most commonly used probes are radioactively labeled probes, which are made of nucleotides with isotope 32. The probe is identified using an X-ray film that is exposed to radiation. The other common probe is the fluorescently labeled probe which emits light under different wavelengths when light shines on it.
DNA probes are made in smaller quantities and then amplified using PCR. The DNA labeling of the fragments either uses radioactive isotopes or a fluorescent dye that glows under certain wavelengths of light. DNA probes can be used to detect mutant alleles and heritable conditions of health risks. The sequence of the mutant allele is determined by DNA sequencing or by finding the DNA sequence in a genetic database. A probe is then made by synthesizing a fragment of DNA that has a complementary base sequence to the mutant allele.
To locate the mutant allele DNA probes and hybridization are used: first, the base sequence of the mutant allele is distinguished and then a fragment of DNA is produced that is complementary to the mutant allele being located. The PCR amplifies multiple copies of the DNA probe. A DNA probe is made by attaching a marker such as fluorescent dye to the DNA fragments. The DNA from the mutant allele is heated to separate the double strands and then it is cooled in a mixture containing many of the DNA probes. If the DNA contains the mutant allele, the probes will bind to the DNA fragments that are complementary in a process called hybridization. The DNA is then washed clean of any unattached probes and the hybridized DNA can be detected because of the presence and intensity of fluorescence. Genetic screening can be used to detect various genetic diseases at the same time. This process is done by fixing hundreds of different DNA probes in an array on a glass slide. The glass slide has holes that hold DNA probes and each of those probes will detect different diseases. These DNA probes are complementary to sequences of DNA that are known mutations that cause genetic disorders. The DNA probes are designed to only fluoresce when they are bound to a DNA fragment by hybridization. Many copies of DNA from the individual that is being tested are then added to the glass slide. If any of the DNA fragments are complementary to a specific probe then it will bind and the probe will fluoresce, indicating the tested individual is carrying that mutation.
Genetic screening can be used to test DNA from cancer cells to detect certain alleles. This allows a targeted therapy for that mutated allele and only specific cancer medication can be used to target the mutation. Another use of genetic screening using DNA probes is to find whether an individual has a gene that puts them at a higher risk of developing a particular kind of cancer. E.g. women with BRCA1 and BRCA2 alleles are more likely to develop breast cancer than other women.
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