Detection of an Alu Polymorphism by Polymerase Chain Reaction

As illustrated below, this exercise has four distinct steps. Since all four steps cannot be completed in one three hour session, the different steps will be completed over the course of several weeks. When working on any given step, it is a good idea to be aware of the overall exercise.

Step one extract cheek cells Step 2 Isolate DNA Step 3 Amplify (PCR) DNA region that may or may not have pv-92 Step 4 electrophoresis to resolve amplified regions, & analyze

Objectives 1. Reinforce concepts of exons, introns, ing genes) present in each human cell. and DNA synthesis The Alu family of transposons are only about 300 base pairs in length. When 2. Gain an insight into what transposons one is “activated”, it makes a copy of it- are, and their number and significance in self, and this copy is inserted randomly the human genome into one of the 46 chromosomes. As might be expected, the number of transposons 3. Learn techniques important to and per cell increases each time one is commonly used in biotechnology. copied. Over millions of years, the num- ber of Alu type transposons has grown to 4. Each student is to determine the num- the extent that each human cell has over ber of copies, if any, of a particular jump- 1,000,000 copies. With so many copies, ing gene he or she has. the Alu type of transposons amounts to approximately 10% of human DNA. Background Information Each human chromosome is a very long Exactly where in a chromosome a trans- double stranded DNA molecule, and con- posable element inserts itself could be of tains millions of nucleotides (A’s, T’s, G’s great consequence. To see how, one and C’s). The pattern of these A’s, T’s, needs to know that most of the 40,000 G’s and C’s form the complete human plus human genes code for proteins. genome. It is currently estimated that the Whether a protein is an enzyme, a trans- human genome has over 40,000 genes. port molecule, or has some other function, While these genes code for the cellular each protein contributes to some aspect activities that collectively result in a hu- of cell life. Most genes have exons (cod- man being, a great part of human DNA, ing regions) and introns (non-coding re- about 25%, belongs to a category called gions). The A’s, T’s, G’s and C’s within transposons. exons code for the amino acids that make up the functional protein. Any change in Transposons, or jumping genes, have no the coding region (exon) of a gene could human related function, they serve only to be disastrous because the change might perpetuate themselves. There are a vari- result in the production of a protein that ety of different types of transposon (jump does not function normally. Severe hu- 69 man diseases, such as mental retarda- Then read over the text giving Alu stats, tion, immunodeficiencies, and cancer, are number of Alu copies per cell, what per caused by changes in the coding regions cent of the human genome Alu occupies, of certain genes. Neurofibromatosis, a tu- how long Alu elements are, and type of mor disease, is an example of a human transposon Alu is an example of. disease caused by the insertion of an Alu transposon into the coding region of a NEXT gene, the NF1 gene. In contrast, inser- Click on MEDIA/ANIMATION tions into introns (non-coding regions of a Then click on “How Alu Jumps” to see gene) generally have no effect on a the jumping mechanism. gene’s protein product. What evidence is there that Alu trans- Since there are so many transposons in posons are “retrotransposons?” every cell, and since insertions into exons can have serious consequences, it is of- The heart of this exercise is that you will ten asked if transposons can have any use state-of-the-art biotechnology to de- benefits. One school of thought is that the termine how many, if any, copies of PV-92 many transposon copies increase the you have. probability of molecular events where seg-  You will first isolate your own ments of DNA from different areas are ex- DNA from a sample of your cheek changed. Because such exchanges can cells. give rise to new genes and new gene  You will then use PCR to make combinations, is thought that transposons millions of copies of a targeted re- might be significant in evolution. gion (the region that may or may not have PV-92) of your genome. Alu-pv92 is the specific transposon  Finally, you will use elec- that is the focus of this exercise. This trophoresis to resolve the DNA insertion is found within an intron of a you made millions of copies of. gene that located in chromosome num- ber 16. Since the Alu-pv92 insertion oc- Polymerase Chain Reaction (PCR) curs within an intron, the insertion has no The web site mentioned above is also ex- effect on the production of this gene’s pro- cellent for its PCR animations. This ani- tein. While the Alu-pv92 insertion is wide mation lets you see how PCR works, and spread in human populations throughout helps reinforce the concepts of how DNA the world, its frequency is greater in cer- strands are held together, what primers tain parts of the world. Nonetheless, it is are and do, and how DNA synthesis is ac- expected that several students in each complished. laboratory section will have one or two copies. Use the following address to go directly to the PCR animations The web site http://vector.cshl.org/geneticorigins http://vector.cshl.org/geneticorigins/pv92/a is very good. It explains what Alu trans- luframeset.htm posons are, how they make copies of themselves, and how the copy inserts it- Click on MEDIA/ANIMATION self elsewhere. Then click on Polymerase Chain Reac- tion First open the web site. Click on the PV-92 insertions icon Answer the following based on the web- Click on Continue on to Alu Insertion site introduction Polymorphisms 70 region that may or may not have PV- What is a primer, and what do they do? 92) What two innovations are important to PCR?

Next press “Menu” (lower left on the screen), then click on “Amplification”

Try to answer the following questions as you proceed through the PCR animation. Be sure to ask your instructor if you can not figure out the answers.

What holds the DNA strands together?

Why is a high temperature required during denaturation?

What happens during the annealing step?

Why must the temperature be reduced during the annealing step?

What happens during the extend primers step?

Note that you can repeat a step many times. This is helpful to reinforce what is going on at a given step.

Press “Go to Second Cycle” and contin- ue until you see the results of the fifth cy- cle.

When finished with the PCR animation, Click on the Menu (lower left on the screen). Then Click on Amplification Graph. Keep clicking on Next Cycle until you have 25 cycles.

How many copies of the targeted re- gion are there after 25 cycles?

Please visit this web site and answer all of the questions before you go lab to do Step three (PCR to amplify DNA . 71 Two very important facts regarding PCR are

1. primers determine the beginning and end of a specific segment DNA to be amplified

2. the number of DNA segments doubles after each cycle (separating DNA strands, primer binding, and extending the primer)

Using PCR to detect the presence of PV-92 So how does PCR allow one to determine if they have one, two, or no copies of the son, and the intron of this person’s other pv-92 transposon? First recall that pv-92 chromosome #16 does not have pv-92. is located in an intron of chromosome #16, and that everyone has two chromo- Arrows show where the primers used in some #16’s (one contributed from their PCR will bind. Primer one will determine mother, and the other one contributed the beginning, and primer two will deter- from their father at the time of mine the end of the intron region that PCR conception). will make millions of copies of.

In the following example both chromo- In this example case, PCR would make some #16’s of an individual is shown. In millions of copies that are 550 bases this case, the intron of one of the person‘s (does not have pv-92), and also millions #16 chromosomes has the pv-92 transpo of copies that are 850 bases (has pv-92). The next step would be to sort out and look at these two different sized pieces.

72 Determining PCR pro duct size one determine the size of the PCR prod- It should be apparent from the two exam- uct? The method used is called elec- ples that the size of the amplified segment trophoresis. First, DNA samples are load- is used to determine the presence of the ed onto a gel, and electric current is ap- 300 base pv-92 transposon. If the ampli- plied. Because DNA is uniformly nega- fied segment is 550 bases long, then it tively charged, DNA is caused to migrate does not contain the transposon. Howev- through the gel toward the positive elec- er, if the amplified segment is 850 bases trode. Shorter molecules migrate faster long, then the amplified segment contains then longer ones. Often DNA of known the 300 base transposon. How then does sizes (a ladder) is run in the same gel.

The following is an example of a gel run in a previous Bio 111 class. Samples were loaded in at the top. A B C

1000

500

100

Discuss the following with others at your table. Which sample B or C contained DNA segments that were shorter? Noting the band of the ladder that is 500 bases, approximately how big is the DNA of sam- ple C? How can you explain the two DNA bands of sample A? Which band A, B, or C is homozygous without the transposon? Explain Which band A, B, or C is homozygous with the transposon? Explain Which band A, B, or C is heterozygous with transposon? Explain

73 Laboratory Procedure As illustrated below, this exercise has four the course of several weeks. When work- distinct steps. Since all four steps cannot ing on any given step, it is a good idea to be completed in one three hour session, be aware of the overall exercise. the different steps will be completed over

Step one Step one extract cheek cells Step two Isolate DNA Step three Amplify (PCR) DNA region that may or may not have pv-92 Step four electrophoresis to resolve amplified regions, & analyze

Cheek Cell Extraction Do not consume food or drink for at least 3. Insert the cotton portion of the swab 30 minutes prior to cheek cell extraction. into the mouth of a 1.5 ml microcentrifuge tube. The, using scissors or a pair of 1. With a sterile cotton swab, gently dikes, cut off the stick just above the cot- scrape the inside of one cheek six times. ton so that the cotton part falls into the Without rotating the swab, move the swab tube. Close the lid, use a water insoluble directly over to the inside of the other ink to label your tube, and place the tube cheek and gently scrape six times. into the rack provided by your instructor. Your instructor will then place the tubes in 2. Gently touch part of the swab contain- the freezer (-20 o) for storage. ing your cheek cells on a clean glass slide once. Add a drop of methylene blue, then 4. Make a drawing of your cheek cells in a cover slip. Examine using the high dry the box provided below, be sure to objective. indicate the magnification.

74 Step two Step one extract cheek cells Step two Isolate DNA Step three Amplify (PCR) DNA region that may or may not have pv-92 Step four electrophoresis to resolve amplified regions, & analyze

DNA Isolation 1. Add 400 l of phosphate buffered saline 7. Add 500l Quiagen buffer AW1 with- (PBS) to the tube containing the cotton out wetting the rim of the spin column. swab coated with your cheek cells. Centrifuge at 8,000 RPM for one minute. This, and the next step serve to wash the 2. Add 400 l of Qiagen buffer AL. This DNA. Since these wash solutions contain contains a detergent to aid in cell disrup- ethanol, the DNA remains precipitated tion, and to solubilize hydrophobic com- and unable to pass through the filter in the pounds. spin column. Discard the tube containing the filtrate, and insert the spin column into 3. Add 20 l of protease K solution. a new 2 ml microfuge tube. Close the lid and vortex immediately for 15 seconds. Immediate mixing is required 8. Add 500 l of Qiagen buffer AW2 with- to maximize cell lysis. This enzyme di- out wetting the spin column rim. Cen- gests proteins, which will aid cells lysis, trifuge at 14,000 for 3 minutes. Complete and in isolating the DNA. removal of the AW2 buffer is necessary as its presence would prevent subsequent 4. Place your microcentrifuge tube in a resolubilization of the DNA trapped in the o heat block set to 56 , and incubate for ten spin column. Therefore, carefully re- minutes. Remove the tube and tap the move the 2 ml microfuge tube to avoid tube on the counter to cause droplets, that splashing the filtrate back on to the spin may have condensed on the inside of the column. Discard the microfuge tube con- lid, to fall into the solution below. taining the filtrate. 5. Add 400 l of pure ethanol (190 –200 9. Insert the spin column into a sterile 1.5 proof). Vortex for 15 seconds. This ml microfuge tube. Add 150 l of AE buf- amount of ethanol will cause the DNA to fer. This buffer has no ethanol and will precipitate but will leave the other com- bring the precipitated DNA back into solu- pounds (proteins, carbohydrates, lipids, tion. Incubate at room temperature for and salts) to remain in solution. one full minute to give the DNA time to dissolve in the buffer. 6. Remove 700 l and place into a QI- Aamp spin column that is seated in a 2 ml 10. Centrifuge at 8,000 RPM for one microfuge tube. Centrifuge at 8000 RPM minute. The collection tube now contains for one minute. At this point the precipi- your isolated DNA in solution. Label the tated DNA is retained by the filter in the tube with a water insoluble marker, and spin column, and the soluble compounds place it in the rack provided by your in- have been forced to the tube below. structor. You instructor will store the tubes at –20oC. Discard the tube containing the filtrate, and insert the spin column containing your DNA into a new 2 ml microfuge tube.

75 Step three Step one extract cheek cells Step two Isolate DNA Step three Amplify (PCR) DNA region that may or may not have pv-92 Step four electrophoresis to resolve amplified regions, & analyze

Polymerase Chain Reaction (PCR) How many cycles is the machine pro- 1. Obtain a PCR reaction tube containing grammed for?______a PCR reaction bead Once the reaction has started, observe 2. Add the following the PCR animation on the computer  10l of your own isolated DNA hooked up to the web! and  15 l of the primer/loading dye mixture Once the program has run its course, your instructor will remove the tray containing 3. Close the PCR reaction tube lid, and all the reaction tubes, and will store them mix the contents. Gently tap the tube on in the in the freezer. the counter to cause all the liquid to go to the bottom of the tube

4. Place you reaction tube into the ther- mocycler, and record its location. Considering that each cell has billions of nucleotides arrayed on 46 chromosomes, Location ______how many places will the primers, shown below anneal to? 5. After your instructor starts the 9700 thermocycler, observe one complete cy- cle. Be thinking about what is occurring at each of the steps in a given cycle. Then, record the temperature for the following:

Denaturation ______Annealing ______Notes: Extension______The PCR reaction beads contain  Taq polymerase, a temperature resistant DNA polymerase,  Mg ions needed by the enzyme,  buffer to maintain the correct pH, and  A, T, G, and C nucleotides.

The primer mixture contains two primers, one for the beginning and one for the end of the region to be amplified. The sequences of these two primers are

5’ AACTGGGAAAATTTGAAGAGAAAGT, and 5’ CTCAAGAAACAGAAGCCCTGTCACC 76 Step four

Step one extract cheek cells Step two Isolate DNA Step three Amplify (PCR) DNA region that may or may not have pv-92 Step four electrophoresis to resolve amplified regions, & analyze

Electrophoresis to resolve amplified regions and analysis Work in groups of 6

1. Following instructions given by your in- structor, prepare a two percent agarose 4. Each student of the group is to load 10 gel as follows: l of their own PCR product into a well. o weigh out 0.40 grams of agarose Since the PCR reaction mix already con- o add to a 50 ml flask containing 20 tains a loading buffer, there is no need to ml of TAE buffer add additional loading buffer to it. o microwave on high for 30 seconds o use tongs, as the flask is now very hot, and swirl the contents to insure 5. Secure the gel tank cover in place, at- all of the agarose is dissolved tach electrical cables, set the power sup- o allow solution to cool before pour- ply to 85 volts (this gives a field strength ing into the gel casting tray. of 5 volts per centimeter), and press the start button. 2. When the gel is solidified, takes about 10 minutes, pour TAE buffer into the 6. When the loading buffer dye is between reservoirs until there is about one millime- half and three quarters across the gel, ter of buffer above the gel turn off the power supply.

3. At least one well per gel is to have a 7. Wearing eye protection and gloves, lift 100 bp ladder. One person of the group the tray containing the gel out of the gel should load 5  l of the ladder (already tank, and carry it to where the ethidium contains a loading buffer) into the center bromide containers are located well. . .

ETHIDUIM BROMIDE IS A CARCINOGEN Gloves and full eye protection must be used whenever working close to ethidium bromide

8. Hold the tray close to the ethidium bro- ethidium bromide solution. Let sit for mide solution. Using one finger, protect- about 10 minutes. During this time, ethidi- ed by gloves, gently push the gel into the um bromide binds to DNA.

77 9. Use a spatula to transfer the gel from tor of the Gel Doc 2000. Your instructor the ethidium bromide solution to water. will point out the orange fluorescence of Let sit an additional 10 minutes. During ethidium bromide bound to DNA, will cap- this time unbound ethidium bromide dif- ture the image, and will print out one copy fuses into the water. This will result in a for each student. Paste the picture of this cleaner, sharper picture. gel next to the gel shown below.

10. Still wearing gloves and eye protec- 11. Use the 100 bp ladder to determine tion, pour out the water from the rinse the size of each DNA band. From the re- tray, and carry the tray containing the gel sults determine your genotype with re- over to the Gel Doc 2000. Use a spatula spect to the Alu-pv92 insert. to transfer the gel on to the trans-illumina-

78 Where is pv 92? As stated earlier in this exercise, pv 92 resides in an intron within a gene that is located on chromosome 16. However, a question students ask is “what is the name of the gene that houses the intron that pv 92 resides in?” This part of the exercise, actually an outside of class assignment, is to answer this question using state-of-the-art tools currently used in in- dustry.

Each chromosome is essentially a very long DNA molecule that contains the bases A, T, G, and C bonded together in a very long chain. Importantly the human genome has been se- quenced, that is from the tip to the end of each chromosome researchers have determined the actual sequence of As, Ts, Gs, and Cs. It has been found that each region of the chro- mosome, each gene, has its own unique sequence of As, Ts, Gs, and Cs. So, if one knew the sequence of a region like an intron, one could simply scan the entire DNA sequence to locate precisely where on which chromosome this sequence is located. This is exactly what you will do. However, there is one problem. You need the sequence of the intron pv 92 resides in. Recently several Cal Poly undergraduate students did this, and the se- quence is posted on a web site. So how do you find where on chromosome 16 this se- quence is located? You will use a data base that contains the human genome. The follow- ing will lead you through the steps.

First, obtain the intron sequence. Visit the following website: www.bio.calpoly.edu/ubl

Click on “protocols,” then click on “Alu sequence.” Copy the entire sequence.

Visit the UCSC web site at: http://genome.ucsc.edu/cgi-bin/hgGateway. Click on “Blat” at the top of the screen

Home - Genome Browser - Blat - Table Browser - FAQ - Help Human Genome Browser Gateway

79 Human BLAT Search

BLAT Search Genome Genome: Assembly: Query type: Sort output: Output type: Human July 2003 BLAT's guess query,score hyperlink Submit Please paste in a query sequence to see where it is located in the genome. Multiple sequences can be searched at once if separated by a line starting with > and the Reset sequence name.

Rather than pasting a sequence, you can choose to upload a text file containing the sequence.

First make sure “July 2003” appears in the Assembly box. Next, click in the box, and then paste in the sequence you copied from Cal Poly’s UBL site.

Then click on the submit button. The site’s software will begin scanning the entire genome, and will stop when it finds a match to the sequence you submitted. It is incredibly fast! When finished the Blats Search results will be displayed.

80 BLAT Search Results ACTIONS QUERY SCORE START END QSIZE IDENTITY CHRO STRAND START END ------browser details YourSeq 453 2 462 468 100.0% 16 - 826917 82692186 browser details YourSeq 21 196 217 468 100.0% 7 + 108763513 108763536

Next, click on “browser” for chromosome 16

The next screen (below) to come up is complicated. For the purpose of this class all we need is the name of the gene, and to know what the gene does. Cick on CDH13. The screen to come up will give you all of this information! Read the summary at the top of the page. Your instructor will later help you understand function of this gene.

Home BLAT DNA Tables Convert Ensembl Map View PDF/PS Guide UCSC Genome Browser on Human July 2003 Freeze <<< << < > >> >>> 1.5x 3x 10x base 1.5x 3x 10x move zoom in zoom out

chr16:82,689,624-82,694,283 620 jump position size 4,660 bp. image width:

Click here to get the name of the gene

Powerful stuff!

81 Review Questions: 1. Indicate the genotype of each lane in the gel below. Also indicate which bands have the Alu-pv92 insert, and which bands do not contain the Alu-pv92 insert.

2. What is lane 6 of the above gel, and how is it used?

3. See the questions on page 73

4. What is a primer, and how is a primer used in PCR?

5. Starting with a gene, make an annotated flow diagram that illustrates transcription, in- tron removal, protein synthesis, and finally function of the protein coded for by the gene.

6. What are the three steps in a PCR cycle, and what do each do?

7. Compared to the amount of DNA at the beginning of one PCR cycle, how much DNA is present at the end of that cycle? How many copies of the DNA region targeted by the primers present after 30 cycles? (see the web site http://vector.cshl.org/geneticorigins)

8. What is an exon, intron, and promoter?

9. Describe the possible effects on the production of a functional gene product (protein) if a transposon was inserted into an exon, intron, or promoter of a gene.

10. What does gel electrophoresis do, and how is this accomplished?

11. Why is it necessary for DNA strands to separate during DNA replication? Compare how the separation of DNA strands occurs during DNA replication in a human cell with how DNA strands are separated in PCR.

12. What does Taq polymerase do in PCR? What is the source of Taq polymerase? Compare the properties of Taq polymerase with human DNA polymerase.

13. The following questions relate to DNA isolation: What was accomplished when cheek cells were incubated with detergents and proteinase K? Why was ethanol added? What purpose did the filter in the spin column serve?

14. If your mother were homozygous with pv-92 and your father was homozygous without, then what pattern on the gel you expect from your DNA? Explain

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