Essay on Using PCR and Gel Electrophoresis to Determine Genotype

PCR is a technique used in laboratories to “amplify a single or few copies of a segment of DNA” (Science Direct, 2017). An example of what can be discovered using PCR is the Alu repeat, which is performed by DNA amplification. The Alu repeat is a “repetitive element of a DNA sequence that is repeated almost 500,000 times throughout the human genome” (Bio-Rad Laboratories, Chromosome 16: PV92 PCR). The Alu sequence is seen in population genetics, which is a “field of biology that studies the genetic composition of biological populations and changes in genetic composition that result from the operation of various factors, including natural selection” (Okasha, 2006). The Alu sequence is seen in population genetics due to the different genetic compositions. Population genetics can be determined using the Hardy-Weinberg principle. The Hardy-Weinberg principle is a “relationship between the frequencies of alleles and the genotype of

Polymerase Chain Reaction was run and the results were placed into an electrophoresis gel to visualize the PCR product. To determine the alleles in the gene, a digest enzyme, Fnu4HI, was placed into the DNA. Tasters would view a cleavage formed at a restriction site in at least one of the alleles, whereas non-tasters would not.

Based on your procedure and results, how is DNA isolated from plant or animal cells? /4

(PCR), which isolates small fragments of DNA that have a high degree of variability from

Four microcentrifuge tubes were placed in a rack, labeled and numbered, in order to identify the group and the DNA/restriction enzyme that it held. Each of the tubes initially received 10 microliters of reaction buffer. There were two samples of suspect DNA provided along with two restriction enzymes (EcoRI and HindIII). Tubes labeled 1 and 2 received 15 μL of DNA from suspect one while tubes 3 and 4 received 15 μL of DNA from suspect two. Following that, 15 μL of Enzyme 1 (EcoRI) were added to tubes 1 and 3, and 15 μL of Enzyme 2 (HindIII) were added to tubes 2 and 4. (Table 1). The tubes were then gently tapped on the counter to mix the DNA and enzyme solution followed by incubation at 37°C for 45 minutes. After incubation, 5 μL of 10x gel loading dye were added to each of the four tubes of suspect DNA. The tubes were then placed on ice while the gel was under preparation.

Requirments: The total reaction volume was 10 μL, containing 1 μL of 10× LightCycler DNA Master Hybridization enzyme mixture (Roche Diagnostics), 3 mM MgCl2, 0.1 μM hybridization probes, and ARMS primers.

However, DNA fragments of 3 lab subjects didn’t show up on the gel. The allele

The DNA extraction results, along with the PCR product, did not fare well. There was not enough product produced to be viable in the later stages of the experiment, so a backup was used in place of the original product.

Following this action, in tubes 1 and 3 15-µl of Enzyme 1 was dispensed and the same amount was dispensed for tubes 2 and 4 of Enzyme 2. The final volumes of the each of the reaction tubes had a final volume of 40-µl (Table 1). After dispensing what was needed in each of the tubes, one of the team members tapped each tube to mix all the contents together and to be sure that the contents were at the bottom of each tube. Sequentially, after all the contents were mixed and were at the bottom, the tubes were then placed in an incubator at 37°C for 45 minutes (Upadhayaya, 2016). Prior to removing the DNA samples from the incubator, the Gel for Electrophoresis had to be casted.

Dr. Park had created the gel for the class. I added 10 μl of the 5x PV92 XC loading dye into the PCR sample. I added 10 μl of the MMR-DNA marker into lane one, 10 μl of the homozygous (+/+) into lane 2, 10 UL of the homozygous (-/-) into lane 3, 10 μl of heterozygous (+/-) into lane 4, and 20 μl of my sample into lane 5. After the other students loaded their sample into the gel, I hooked up the electrodes and turned on the electrophoresis apparatus, which was at 100 volts. The electrophoresis apparatus ran for 40 minutes, which was until the marker dye had traveled half of the distance to the end of the gel. I then removed the gel and submerged it in the Fast Blast DNA stain (100x) using the staining tray. To create the staining gel, we used the formula M_1 V_1= M_2 V_2. The initial concentration of the gel was 500x, but we needed 100 ml of a concentration of 100x. I calculated that we needed 80ml of H_2 O and 20 ml of the dye. I stained the gel for 2 minutes. I rinsed the gel in warm water for 10 minutes. I compared my sample lane with the control lane and recorded my

In which 50 µl of PCR master mix used for amplification of ITS1 region .

This is useful for pathogens that are difficult to culture, or where such culturing is potentially hazardous. The amplified products of PCR, however, are not immediately identifiable, and must be put through another process known as gel electrophoresis before data can be analysed. Gel electrophoresis involves the separation and analysis of DNA (or many other macromolecules) based on size and charge. Nucleic acid molecules are separated by applying an electric current to move negatively charged strands through a solution of agarose gel. Shorter fragments move faster and migrate further than longer fragments, meaning the base length of an unknown fragment can be measured against a known fragment in order to identify it. This is seen through coupling the DNA with a fluorescent tag (marker).

Mullis new method is so significant, he was awarded the Nobel Prize. Polymerase chain reaction is a four step highly specific and rapid method, that can produce billions of copies of DNA, within a test tube in a small amount of time. This process, outlined below, will repeat and as more cycles are completed, the amount of DNA produced will increase as well. The first step in the PCR chain, is the DNA sample itself being heated to the high temperature of 95℃, resulting in the denaturation of the double-stranded DNA, into single-strands. The DNA using two nucleotide primers, is cooled to an approximate temperature of 55℃. Each primer are compatible to each 3’ end of the DNA fragments to magnify it. The reason for the cooling temperature, is because it allows the primers to anneal, with the 3’ end of the single stranded DNA to also be magnified. The third step, in this process, requires the sample to be heated again, but to a slightly less temperature of 72℃, which Mullis found to be the optimal temperature for Taq polymerase, which is a specific kind of DNA polymerase used for the PCR. Without the isolation of the DNA polymerase from the heat-loving bacteria, which ultimately makes PCR able to work. The same as different DNA polymerase enzymes, by adding a nucleotide to the ends of the DNA(using base pairing), it synthesizes. Like stated before, the

PCR (Polymerase Chain Reaction) is a laboratory technique that takes specific fragments of DNA and amplifies them using primers and a polymerase that can withstand high temperatures. The materials needed to complete this experiment include the DNA fragments that are to be amplified, two primers (one to attach to the top strand of the DNA fragment of interest and one to attach to the bottom strand on the other end), Taq DNA polymerase, dNTP mix, MgCl2, PCR buffer and the PCR machine. Primers are necessary for the Taq DNA polymerase to attach to in order to begin copying because primers can only attach to an existing piece of DNA. Taq DNA polymerase is necessary for PCR rather than DNA polymerase found in our bodies because when the DNA fragments of interest are heated in order to denature them and separate the two complementary strands, DNA polymerase also denatures. Taq DNA polymerase is taken from a strain of bacteria (Thermus aquaticus) that is taken from the hot springs of Yellowstone National Park, and it can withstand boiling temperatures which makes it effective for PCR. dNTP mix is a mixture of nucletotides that the polymerase uses to create the new

The polymerase chain reaction or PCR for short can be used to create many copies of DNA. This allows the DNA to then be visualized using a dye like ethidium bromide after gel electrophoresis. The process has been refined over the years, however the basic steps are similar.