DNA Gel electrophoresis & PCR Worksheet-Lydia Specht

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Biology

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Apr 3, 2024

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PCR and DNA Gel Electrophoresis 10 Points Application of PCR and DNA gel electrophoresis in diagnostics : Chronic Hepatitis is a condition of the liver that is caused by many different viruses. Of these, Hepatitis B (HBV) and Hepatitis C (HCV) have been identified as the top causes of hepatitis. Hepatitis B, C and D are very similar in their modes of transmission and are spread by contact with infected blood and contaminated syringes. Simultaneous co- infections with B and C can occur in patients. A group of patients (1-5) was tested for Hepatitis C which has an RNA genome. Briefly blood samples were taken and RNA was extracted. RNA samples were subjected to amplification by RT-PCR. The products were resolved by agarose gel electrophoresis. The picture of the gel is shown below in Figure 1. L is the DNA standard; - and + are the negative and positive controls respectively; 1-5 are the RT-PCR amplified samples from 5 patients. A positive amplification of HCV and HCB are indicated by arrows. Next, the blood samples from the same patients was used to screen for HBV, which has a DNA genome. Briefly, blood sample was subjected to PCR to amplify the HBV gene and then resolved by agarose gel electrophoresis. The picture of the results is shown in Figure 2 below.

1. (2 pt) What is the principle behind agarose gel electrophoresis? The principle behind agarose gel electrophoresis includes an electric field and with a gel and microscopic holes, molecules are then pushed apart by the electric field. The total length of the molecule has an inverse relationship with the rate at which they pass through the pores in the gel. 2. (2.5 pt)Which patients are infected with HCV, HCV and both HBV and HCV (co- infection) and no infection? How did you make this conclusion? Patients #1, 3, and 5 are positive testing and patients 2 and 4 are negative testing. In the patients 2 and 4, there is no disease at all. I made this conclusion based on the test results above and for the indicative markers signaling positive testing. 3. (1.0 pt) Using points A and B as reference, predict the direction that the DNA molecules moved in the gel. Did they move from A to B or from B to A? (Hint: look at the sizes of the standards in lane L) DNA molecules move from larger to smaller sizes so in this case it would be DNA molecules move from A to B. 4. (2.5) Briefly explain the steps that were done in Figure 2 to amplify the viral nucleic acid (you need not explain the agarose gel electrophoresis part) . How did this differ from the amplification in Figure 1? There is a 2 step procedure in RT-PCR which is more sensitive than the one-step procedure. -Put in a PCR tube, template RNA, Primer, dNTP mix and nuclease free water. -Then include the reverse transcriptase and RNase inhibitor. -Add that in to the PCR tube

-Then put the PCR tube in one round of a heat cycle, which would also include annealing. The reverse transcriptase is then activated and then extended. Immediately do PCr or store on ice untik you can do PCR. All PCR tubes should have the mix, buffer, dNTP, MgCl, Taq polymerase and nuclease free water. Which should also include the proper primer. Amiplification procedure includes 3 steps 1. Denaturation 2. Annealing (PCR in a thermal cycler) 3. Elongation. 5. (2.0 pt) What is the purpose of negative and positive controls in this experiment? The “positive controller” in this case is used to validate the point of the experiment. The negative control in the experiment does not respond to the test, and is used to determine how influences can alter the test results.

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