Techniques in molecular genetics

Techniques in molecular genetics
There are three general techniques used for molecular genetics: amplification, separation and detection, and expression. Specifically used for amplification is polymerase chain reaction, which is an “indispensable tool in a great variety of applications”[2]. In the separation and detection technique DNA and mRNA are isolated from their cells. Gene expression in cells or organisms is done in a place or time that is not normal for that specific gene.

Amplification
There are other methods for amplification besides polymerase chain reaction. Cloning DNA in bacteria is also a way to amplify DNA in genes.

Polymerase chain reaction
The main materials used in polymerase chain reaction are DNA nucleotides, template DNA, primers and Taq polymerase. DNA nucleotides are the base for the new DNA, the template DNA is the specific sequence being amplified, primers are complementary nucleotides that can go on either side of the template DNA, and Taq polymerase is a heat stable enzyme that jump-starts the production of new DNA at the high temperatures needed for reaction. This technique does not need to use living bacteria or cells; all that is needed is the base sequence of the DNA and materials listed above.

Cloning DNA in bacteria
Main article: Molecular cloning
The word cloning for this type of amplification entails making multiple identical copies of a sequence of DNA. The target DNA sequence is then inserted into a cloning vector. Because this vector originates from a self-replicating virus, plasmid, or higher organism cell when the appropriate size DNA is inserted the “target and vector DNA fragments are then ligated”[2] and create a recombinant DNA molecule. The recombinant DNA molecules are then put into a bacteria strain (usually E. coli) which produces several identical copies by transformation. Transformation is the DNA uptake mechanism possessed by bacteria. However, only one recombinant DNA molecule can be cloned within a single bacteria cell, so each clone is of just one DNA insert.

Separation and detection
In separation and detection DNA and mRNA are isolated from cells (the separation) and then detected simply by the isolation. Cell cultures are also grown to provide a constant supply of cells ready for isolation.

Cell cultures
A cell culture for molecular genetics is a culture that is grown in artificial conditions. Some cell types grow well in cultures such a skin cells, but other cells are not as productive in cultures. There are different techniques for each type of cell, some only recently being found to foster growth in stem and nerve cells. Cultures for molecular genetics are frozen in order to preserve all copies of the gene specimen and thawed only when needed. This allows for a steady supply of cells.

DNA isolation
DNA isolation extracts DNA from a cell in a pure form. First, the DNA is separated from cellular components such as proteins, RNA, and lipids. This is done by placing the chosen cells in a tube with a solution that mechanically, chemically, breaks the cells open. This solution contains enzymes, chemicals, and salts that breaks down the cells except for the DNA. It contains enzymes to dissolve proteins, chemicals to destroy all RNA present, and salts to help pull DNA out of the solution.

Next, the DNA is separated from the solution by being spun in a centrifuge, which allows the DNA to collect in the bottom of the tube. After this cycle in the centrifuge the solution is poured off and the DNA is resuspended in a second solution that makes the DNA easy to work with in the future.

This results in a concentrated DNA sample that contains thousands of copies of each gene. For large scale projects such as sequencing the human genome, all this work is done by robots.

mRNA isolation
Expressed DNA that codes for the synthesis of a protein is the final goal for scientists and this expressed DNA is obtained by isolation mRNA (Messenger RNA). First, laboratories use a normal cellular modification of mRNA that adds up to 200 adenine nucleotides to the end of the molecule (poly(A) tail). Once this has been added, the cell is ruptured and its cell contents are exposed to synthetic beads that are coated with thymine string nucleotides. Because Adenine and Thymine pair together in DNA, the poly(A) tail and synthetic beads are attracted to one another, and once they bind in this process the cell components can be washed away without removing the mRNA. Once the mRNA has been isolated, reverse transcriptase is employed to convert it to single-stranded DNA, from which a stable double-stranded DNA is produced using DNA polymerase. Complementary DNA (cDNA) is much more stable than mRNA and so, once the double-stranded DNA has been produced it represents the expressed DNA sequence scientists look for.