The purpose of today s lab is to:

Transcription

1 Cancer Gene Detection Instructions The purpose of today s lab is to: gain an understanding of the p53 tumor suppressor gene and its role in familial cancers; analyze p53 mutations from normal and tumor cells by electrophoresis; stain the gels; and observe the banding patterns on the gels and use this information to identify p53 mutations. The familial pedigree that you designed in the prelab activity strongly suggests that Li-Fraumeni syndrome runs in Valerie s family. If a genetic counselor (who would be the person to develop the pedigree) uncovered this possibility, he/she would order a secondary diagnostic test to conducted. In this scenario, Valerie provides a sample of blood and tumor tissue to conduct DNA analysis on the p53 gene. Normally, the region of Valerie s DNA with her p53 gene would be amplified using PCR (polymerase chain reaction). Afterwards, one of several methods could be used to detect the presence of a point mutation at the hot spots in her p53 gene. In the simulation experiment which follows, Valerie's DNA has already been digested with a restriction enzyme that recognizes the mutant sequence at the hot spot site at nucleotide 165 which is the palindrome CAGCTG. Because only the mutated p53 contains this sequence, the enzyme will only cut mutated DNA and will leave the normal DNA sequence alone. The predigested samples with the control wild type and DNA markers will be separated by agarose gel electrophoresis and stained. normal p53 mutated p53 amplify region of DNA by PCR amplify region of DNA by PCR mutation treat with restriction enzyme treat with restriction enzyme normal p53 does not have restriction enzyme site; DNA is not cut restriction enzyme recognizes mutated p53 sequence and cuts DNA into two fragments Separate fragments by gel electrophoresis Each normal chromosome should produce one large band. Each mutated chromosome should produce two smaller bands. 111

2 Procedure 1. Obtain a prepared agarose gel. 2. Place the casting tray with the solidified gel in it, into the platform in the gel box. The wells should be at the ( ) electrode end of the box, where the black lead is connected. 3. Ask your instructor to pour ~275 ml of electrophoresis buffer into the electrophoresis chamber. He will pour buffer in the gel box until it just covers the gel and enters the wells. 4. Using a separate pipette tip for each sample, load your digested DNA samples into the gel. Gels are read from left to right. The first sample is loaded in the well at the left hand corner of the gel. Your instructor will do the first on for you so you can see it is done properly. Lane 1 DNA Size Ladder clear tube 40 µl! loaded Lane 2 Control DNA green tube 40 µl! loaded Lane 3 Valerie s peripheral blood DNA blue tube 40 µl! loaded Lane 4 Valerie s breast tumor DNA orange tube 40 µl! loaded Lane 5 Valerie s normal breast tissue DNA purple tube 40 µl! loaded 5. Secure the lid on the gel box. The lid will attach to the base in only one orientation: red to red and black to black. Connect electrical leads to the power supply. 6. Turn on the power supply. Set it for 100 V and electrophorese the samples for min. While you are waiting for the gel to run, you may begin the review questions on the following page. While you are waiting for your gel to finish, answer Question 1 on the Lab Report. 7. When the electrophoresis is complete, turn off the power supply and remove the lid from the gel box. Carefully remove the gel tray and the gel from the electrophoresis chamber. Place the gel and casting tray on a paper towel. Be careful, the gel is very slippery! Proceed to the next section for detailed instructions on staining your gel. Activity 2. Staining DNA with Fast Blast DNA Stain WARNING Although Fast Blast DNA stain is nontoxic and noncarcinogenic, latex or vinyl gloves should be worn while handling the stain or stained gels to keep hands from becoming stained blue. Take special care not to get the stain on your clothes! 112

3 Procedure 1. Stain gels To remove each gel from the gel tray, carefully slide it into the staining tray. Pour approximately 120 ml of 100x stain into the staining tray. If necessary, add more 100x stain to completely submerge the gels. Stain the gels for 3-4 min, but not for more than 4 min. Using a funnel, pour the 100x stain back into the storage flask and save it for future use. The stain can be reused at least 7 times. 2. Rinse gels Transfer the gels into a large container containing ml of clean, warm tap water. Gently shake the gel in the water for ~10 secs to rinse. 3. Wash gels Transfer the gel into a large container with ml of clean, warm tap water. Gently swish the gels in the water once every minute. Students usually find that this works best by simply standing at one of the sinks in the lab and gently flooding the gel with warm tap water continuously. 4. Wash gels Perform a second wash as in step Record results Pour off the water and examine the stained gels for expected DNA bands. The bands may appear fuzzy immediately after the second wash, but will begin to develop into sharper bands within 5 15 minutes after the second wash. This is due to Fast Blast stain molecules migrating into the gel and binding more tightly to the DNA. Answer Questions 2-5 on the Lab Report. 113

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5 Cancer Gene Detection Lab Report Name 1. In the gel below, sketch out the bands that you would expect to see from a DNA sample obtained from tissue... homozygous for the normal p53 (a) homozygous for the mutated p53 (b) heterozygous (c) (a) (b) (c) 2. Why does Valerie's tumor DNA sample have fewer bands than the peripheral blood? (Note: peripheral blood simply refers to a blood sample taken from a vein, similar to what happens when you donate blood.) 3. Can you tell if Valerie is normally homozygous or heterozygous for mutated p53? Is this different in her breast tumor tissue? 4. What is the purpose of the DNA in the control lane? 5. Can a physician proceed with diagnosis based on molecular biology data? 115