Dna Analysis

PV92: DNA Analysis

PCR Amplification and Gel Electrophoresis

Deoxyribonucleic acid is a very important molecule for supporting genetic variation among species. It is a double-stranded molecule held together by weak bonds between base pairs of nucleotides (adenine, guanine, cytosine and thymine), and it encodes genetic information [1]. A large amount of introns (intergenic DNA) can be classified as "non-coding," and much of it consists of repeated segments [2]. One of these segments is the element Alu. It copies itself about 500,000 times, so it constitutes roughly 5% of the human genome [2]. This DNA segment is extremely important, and that is why we want to know as much about it as we can, and someday come to a full understanding of just how important it actually is in our lives.

This experiment was conducted to show that our genotype is determined by our DNA, and going through the experimental process helps with the understanding of how scientists actually obtain certain genetic information. This experiment is an excellent way to see the processes of PCR amplification, electrophoresis, and staining, and to learn how each process works with the other to supply a final outcome. The objectives of the research were to familiarize us with these processes and to give us a greater understanding of how important DNA is and how it is a part of our lives.

In this experiment, DNA from a hair follicle was subjected to a number of processes and then analyzed to find a specific genotype (+/+, +/-, -/-). The class data was then used to perform a chi square analysis, and that was compared to another class's data. This helps in grasping the concept of how important DNA is, and it shows that it does, in fact, exist and fulfills a purpose. Its purpose is generally to ensure genetic variation in a species, therefore ensuring the survival of that species, generation after generation. The nature of this phenomenon will favor some, while others are less fortunate. Overall, DNA is the driving force behind genetics, and we could not survive without it. It is also very helpful to understand how DNA works in certain fields of science. It can be used to catch criminals, improve crops, and especially to detect certain diseases. These things are fairly new concepts, being that DNA was discovered fifty years ago and has only been studied since then, yet they are incredible advancements, and more are sure to come.

Materials and methods

Materials

Two human hairs (with noticeable sheaths and bulbs attached), one small screwcap tube, 200 µl of InstaGene matrix plus protease, scissors, a micro test tube holder, a vortexer, a 56Ñ"C water bath, a 100Ñ"C water bath, a centrifuge, a DNA template, individual deoxynucleotides, DNA polymerase, magnesium ions, oligonucleotide primers, a salt buffer, a thermal cycler, 10 µl of PV92 XC loading dye, an agarose gel, 275 ml of electrophoresis buffer, an electrophoresis chamber, Fast Blast DNA stain [3].

Methods

Part I: DNA Template Preparation

First, we obtained a small screwcap tube with 200 µl of InstaGene matrix. Then we collected two hairs from ourselves, and placed them into the screwcap tube. We incubated the tube for ten minutes in a 56Ñ"C water bath. After five minutes, we removed them from the water bath and vortexed them, and then placed them back into the water bath for another five minutes. After five minutes, we removed our tubes, vortexed them a second time, and incubated them in a boiling water bath (100Ñ"C) for five minutes. We removed our tubes from the boiling water bath, vortexed them for a third time, and centrifuged them for five minutes at 6,000 x g. After this part of the experiment, we refrigerated our tubes for one week [3].

Part II: PCR Amplification

First, we took our tubes out of the refrigerator and centrifuged them for two minutes at 6,000 x g. We then obtained a PCR tube and a capless micro test tube. Next, we transferred 20 µl of our DNA template into the PCR tube, which sat in the capless micro test tube. After this procedure, we transferred 20 µl of the PCR master mix into the PCR tube, mixed the contents of the tube, and tightly capped the tube after doing so. Then we placed the PCR tube into a thermal cycler, where they underwent about 40 cycles of amplification in three hours [3].

Part III: Gel Electrophoresis and Staining of Agarose Gels

To start, we removed our PCR tubes from the thermal cycler and placed them in a micro test tube holder. We placed the PCR tube in a capless micro test tube and put it in a centrifuge to pulse-spin it for three seconds at 2,000 x g. We then added 10 µl of PV92 XC loading dye to our PCR tubes, and gently mixed the contents. Next, we obtained pre-made agarose gels, and placed them on a casting tray on the platform of a gel box. We filled the electrophoresis chamber with 275 ml of electrophoresis buffer, and proceeded to load samples of our templates into 7 wells of the gel in the following order (from right to left): MMR (standard), homozygous (-/-) control, homozygous (+/+) control, heterozygous (+/-) control, Student 1, Student 2, Student 3. Next, we secured the lid on the electrophoresis chamber, attached the electrical leads to the chamber and to the power supply, and set it to 100 V for thirty minutes. When the electrophoresis was complete, we shut off the power supply, removed the lid from the gel box, and removed the gel tray and the gel from the gel box. After that, we slid the gel onto a tray and poured the Fast Blast DNA stain over it, letting it sit there for two to three minutes. Finally, we removed the gel from the stain and rinsed the gel in a large container with warm tap water for five minutes, gently rotating the container the entire time [3].

Results and discussion

The results of this experiment basically showed that my genotype is homozygous negative (-/-). This means that the PV92 sequence does not show up on neither of the two alleles of chromosome 16. Therefore, my DNA band is 641 base pairs long. If my results showed that my genotype was homozygous positive (+/+), the PV92 sequence would have appeared on both the alleles of chromosome 16, and my DNA band would have been 941 base pairs long. If I found that my genotype was heterozygous (+/-), the PV92 sequence would have only appeared on one of the alleles of chromosome 16, and I would have had two different bands of DNA, one that was 941 base pairs long and the other that would be 641 base pairs long.

After the experiment

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