PCR and Electrophoresis

PCR and ELECTROPHORESIS Molecular biology analysis techniques are one of the keys to understanding the central dogma in biology. The existence of this technique will help us understand the processes of replication, transcription, translation, etc. This technique allows us to understand organisms at the molecular level, namely the DNA, RNA, amino acid and protein levels. The technique that will be discussed in this practicum is PCR (Polymerase Chain Reaction).PCR is a technique for synthesizing and duplicating DNA and RNA in vitro. PCR was first developed by Karl Mullis in the mid-1980s. PCR is based on exploiting the ability of DNA polymerase to synthesize new strands of complementary DNA from DNA templates. The mechanism that occurs during PCR is the same as DNA replication in vivo. The PCR process occurs over several cycles. The PCR method can increase the number of DNA sequences thousands or even millions of times from the original number, around 106-107time The components needed in the PCR reaction are DNA template, primers (oligonucleotides), and PCR mix. DNA template is a number of DNA samples that will become a template for the formation of new DNA strands during the amplification process using the PCR technique. A primer is a short sequence of DNA or RNA (oligonucleotide) that is complementary to the template DNA strand used to initiate DNA amplification. PCR mix contains PCR buffer, Mg2+, dNTP, and polymerase enzymes. 1. Working Principle of PCR The working principle of PCR is a repeated reaction carried out by the polymerase enzyme, which is an enzyme that is capable of assembling DNAbuilding blocksinto long molecular strands (Degenet al., 2006). The PCR process involves several stages, namely: (1) predenaturation of template DNA at a temperature of 94-95°C; (2) denaturation (separation of double strands) of template DNA at 94-95°C; (3) attachment of the primer to the template (annealing) at a temperature of 48-60°C; (4) primary elongation (extension) and (5) stabilization (final-extension) at a temperature of 72°C. Stages (2) to (4) are repetitive stages (cycles), where in each cycle the amount of DNA is amplified. Double-stranded DNA template (unamplified DNA) is separated by thermal denaturation and then cooled to a certain temperature to give the primer time to set (annealing primers) in the area 2. Factors that influence the success of PCR The success of primer amplification using isolated genomic DNA as a template is largely determined by the suitability of the primers (composition and composition).primer size) against template DNA and optimization of PCR conditions. PCR Optimizationincludes setting the denaturation temperature and temperature and time of primer attachment (annealing) on the template DNA. The denaturation temperature needs to be optimized, because if denaturation does not occur then the subsequent process will experience errors, both errors in primer attachment and errors in amplification of the desired target. Apart from denaturation, the primer sticking temperature is also a determinantamplification success. Determination of temperatureannealingcan be based on valuemelting temperature(Tm) or the melting temperature of the primer used for amplification. 3. Analysis of PCR Results PCR results can be analyzed after electrophoresis. Electrophoresis is a method of separating molecules that uses an electric field (electro). This method is very commonly used to separate molecules that are charged or made charged. This technique can be used to utilize the electrical charge that exists in molecules, for example DNA, which is negative. Molecules that can be separated include DNA, RNA, or protein. If a molecule with a negative charge is passed through a medium, for example agarose gel, then an electric current flows from one pole to the pole with the opposite charge, then the molecule will move from the negative pole to the positive pole. The results of electrophoresis can be observed with ultraviolet light, which will appear as ribbons on the gel. These bands are DNA molecules that move along the gel after electrophoresis. The following is an example of DNA electrophoresis results to determine the biological father of baby A: 4. PCR application The PCR technique has been widely applied, including for: - nucleotide sequence amplification,detection of infectious pathogens and identification of mutations in genes related to disease risk factors, such as thalassemia screening using PCR -SSCP - field of forensic medicine, - tracing a person's origins by comparing DNA finger prints.