BioMolecules : Applications of Recombinant DNA

Human Cells with a level of assessment for Laws. Just give the analysis of DNA, RNA and others.

Central Dogma of Molecular Biology
DNA (gene)  ->  RNA (transcript)  ->  Protein (trait)

Different organisms have different traits based on their genes (DNA sequences). For example, frogs have antimicrobial peptides on their skin. Some jellyfish have proteins that allow them to glow in the dark. Mutations in hemoglobin genes lead to anemia.

Based on the central dogma, if transcription and translation of genes lead to some traits, then the insertion of certain genes in a given organism may provide it with new traits. This is the basis for the development of genetically modified organisms (GMOs).

Presentation of Recombinant DNA

There are many different traits that can be introduced to organisms to change their properties. The following table shows examples of modified traits using cloned genes and their applications:

PCR Amplification

Once a desired trait is chosen, information must be acquired for either its detection or expression in a given organism.

Detection
Some researchers may be interested in determining if a given gene/trait is available in a particular organism. If no previous research provides this information, researchers may test the DNA of different organisms for the presence of these specific genes. A technique that allows the detection of specific genes in target organisms is called PCR.

PCR amplification is an in-vitro method that simulates DNA replication in vivo. It utilizes a thermostable (heat-resistant) DNA polymerase that builds single stranded DNA strands unto unwound DNA templates. PCR uses repeated cycles of incubation at different temperatures to promote the unwinding of the DNA template (~95°C); the annealing of a primer (a ~20bp oligonucleotide sequence (recall RNA primers in DNA replication) onto the ssDNA template strand (~54 - 60°C); and the extension of the generated ssDNA strand through the binding of complementary bases to the template strand (~72° C).

The thermostability of the polymerase allows it to survive the repeated cycles of denaturation, annealing and extension with little loss of enzyme function. Each cycle of PCR doubles the amount of the target sequence. A typical PCR experiment uses about 35 cycles of amplification. This increases the original amount of the target sequence by 235 (i.e. ~34 billion) times. Gene detection by PCR involves the design of primers that would only bind to sequences that are specific to a target. For example, researchers would want to find out if gene X (e.g. the gene for insulin) is available in a target organism (e.g. a mouse, Mus musculus). Primers may be designed by looking at the available sequences for gene X in the databases (e.g. all the genes for insulin in different organisms; humans, pigs, cows, etc.).

The different gene X sequences must be aligned/ compared to match areas of sequence similarity (conserved sequences) and areas of sequence dissimilarity (non-conserved sequences). Primers designed to have the same sequence as the conserved areas will be specific for binding gene X sequences in all the target organisms. Primers designed to have the same sequence as the non-conserved areas will only be specific for the organisms which match its sequence.

Primers may be classified as forward or reverse primers. Forward primers are complementary and bind to the reverse complementary (non-coding) sequence of the gene. Reverse primers are complementary and bind to the coding sequence of the gene.

PCR Applications

1. PCR may be used to detect the presence of a desired gene in an organism. Depending on the primer design, the expected product may represent only a specific region of the gene or the entire gene itself. The first case is useful for detection of the gene, or the detection of organisms with that specific gene within a sample. The second case is useful for the amplification of the entire gene for eventual expression in other organisms. The direct amplification/copying of a full gene is part of the process for “cloning” that gene.

2. Cloning and Expression
Some genes provide economically, and industrially important products (e.g. insulin-coding genes; genes for collagen degradation). In some cases, scientists would want to put these genes into organisms for the expression of their products. One example would be the insertion of an insulincoding gene from the human genome into bacteria. This allows the “transformed” bacteria to now produce human insulin as a product.

Certain types of bacteria are capable of this process since they are able to take genes within their cell membranes for eventual expression. The genes are normally in the form of small, circular DNA structures called plasmids.

The genes found in the inserted plasmid DNA sequence will be expressed as proteins that provide specific traits to the transformed bacteria. The basic components of an expression plasmid are listed in the following table. The purpose of each of these is also provided.

There are certain ethical principles should be followed and adhered to in the production of genetically modified organisms. Animal welfare should be taken cared of and human cloning must never be conducted.

Lesson 1 - BioMolecules : Structure and Functions
Lesson 2 - BioMolecules : DNA Replication and Protein Synthesis
Lesson 3 - BioMolecules : Genetic Engineering