DNA sequence analyses fall 2023 (1)

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University of Virginia *

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2010

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Biology

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Feb 20, 2024

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Uploaded by DoctorFlower13382

PCR and DNA sequence analysis, fall 2023 page 1 of 9 Introduction As you know, each of you had a choice of two different DNA targets that you could attempt to amplify by PCR and then sequence. Both the PCR and sequencing were successful for most of you (a little more than half of nearly 700 samples sent for sequencing). But for many students there was a problem at some point. To ensure that each of you has the same opportunity to earn full credit, each of you will analyze several samples that I’ve identified as providing representative results in addition to your own. This is a significant graded lab assignment in which all students can earn full credit, regardless of success or failure of PCR or sequencing of their own DNA sample. While I intentionally selected innocuous targets for our work, it should be obvious that by simply using different primers we could determine genotype, and thus predict phenotype, for much more significant gene sequences. For example, a BRCA1/2 mutation would indicate a very high risk of breast cancer, an extended repeat at the Huntingtin gene locus would indicate that future Huntington’s disease was certain, and particular alleles of other genes would demonstrate carrier status for diseases such as sickle cell anemia, cystic fibrosis, etc. DNA gel electrophoresis Following the attempt at PCR amplification (and before sequencing), gel electrophoresis provides a simple, rapid way to obtain evidence of successful PCR. Specifically, in analyzing your DNA agarose gel following electrophoresis we were interested in determining three things: 1) Did you amplify DNA? 2) Did you likely amplify the intended target? 3) Did you amplify more than the intended target? DNA amplification will be evidenced by seeing one or more bands on your gel where the DNA is stained by GelRed. Evidence for amplifying the intended target will come from determining the expected length of your PCR product and finding a band on the gel containing DNA of that length. Determining the expected length of your PCR product On the final two pages of this document, you’ll find the sequences of the primers that we used for PCR and known human DNA sequences in the regions we attempted to amplify (only one strand is shown— we know the other strand will be complementary and antiparallel). Other than at a few bases defining allelic variation, we all have these same human sequences and the PCR primers used should work for all of us. Sequencing will allow identification of allelic variations. For the target you selected, search the provided sequence for a match to the primers. The first primer will show a direct match in the strand shown (I recommend highlighting or underlining the matching region). Being identical to the strand shown, this primer is complementary to the strand not shown (and to which it will anneal in PCR). The second primer is not identical to the strand shown here but rather complementary and anti-parallel (as it will anneal to this shown strand during PCR). A simple way to find this region of the strand shown is to search for the reverse-complement of the second primer sequence. You can use the following tool to determine the reverse-complement of that or any sequence https://www.bioinformatics.org/sms/rev_comp.html Once you’ve identified the primer regions within the target sequence, you can determine the expected length of the PCR product by simply selecting that sequence representing the amplified product and using the word/character count tool in Word or any other text editor. Remember that in PCR we use chemically synthesized DNA primers—being extended, the primers are part of the product. Be careful to avoid inadvertently editing this document. If you have concerns that you may have, simply download a new copy from our Canvas site.

page 2 of 9 Question 1: For the target you selected (TAS2R38 or HERC2) what is the expected product length using our primers? Remember, you’ll be answering these same questions in the associated Canvas assignment “Lab 12: DNA sequence analyses” (within the Quizzes tool) to receive credit. Determining the actual length of your PCR product To determine the length of the DNA molecules in any band on a gel, we compare the migration of those molecules to the migration of DNA molecules of known sizes in one lane of the same gel (referred to as “markers” “standards” or a “ladder”). These known-size DNA standards in one lane of your gel form a 100-base pair (bp) ladder—i.e., DNA molecules that are 100bp long, others that are 200bp long, others of 300bp, etc. up to 3000bp, with a much higher quantity of the 1000bp molecules for easy identification of that band. This ladder is shown below. Given how close in size the standards are, and the limited separation of them on our gels, it’s reasonable to simply “eyeball it” to assess whether you likely amplified the intended target. Question 2: Based on your answer to question 1 with the target you selected, at what position would you expect to see a band containing your amplified DNA? (A-J)

page 3 of 9 DNA sequencing You’ll recall that at lab you added a 10 μ l aliquot of your purified PCR material to one well of a 96- well plate. Your DNA serves as the template for the sequencing reaction. A primer (one of the same two primers that you used for PCR) had already been added to that well. If you are heterozygous in the region sequenced, the DNA template strands will differ at one or more location, resulting in sequencing products of a particular length ending in either of two different dideoxy nucleoside triphosphates (ddNTPs). As shown below at position 287, this results in 2 different colored overlapping peaks, but only at the location of these single nucleotide polymorphisms (SNPs). Note near the middle of this chromatogram the overlapping blue ( C ) and red ( T ) peaks. The base- calling software tries to “guess” which is correct and indicates only that one base, but we know that both are correct and simply reflect heterozygosity. See page 427 in your text for more on SNPs. This is assigned reading relevant to exam 5. There are only a few (1-3) SNPs within our selected targets, and so such overlapping peaks should be infrequent. If you observe frequent overlapping peaks, then either the PCR material contained a mix of different amplified regions of a student’s genome (i.e., multiple different templates) or there was an experimental error, such as cross-contamination resulting in a mixture of different primers in one DNA sequencing reaction. As illustrated above, you will have to look carefully for the presence or absence of overlapping peaks at the location of these known SNPs. Tiny overlapping peaks (like at position 284) are common “noise” in the chromatogram and do not indicate heterozygosity. Heterozygosity is expected to result in approximately equal amounts of the two different sequencing products thus the peaks will overlap closely, and both will be notably lower than surrounding peaks (as illustrated above at position 287). To view the chromatograms, you may download free software (FinchTV for Mac or PC) from our Canvas Files folder. If your Mac won't let you open iFinch because it's from an unidentified developer, here’s how to get around that*: 1. In the Finder on your Mac, locate the FinchTV app that you downloaded from Canvas. Don’t use Launchpad to do this. Launchpad doesn’t allow you to access the shortcut menu. 2. Control-click the app icon, then choose Open from the shortcut menu. 3. In the new window that comes up click Open. The app is saved as an exception to your security settings, and you can open it in the future by double- clicking it just as you can any registered app. *https://support.apple.com/guide/mac-help/open-a-mac-app-from-an-unidentified-developer-mh40616/mac After downloading and installing FinchTV from our course Canvas site, if double-clicking a chromatogram file (all of which have the extension .ab1) doesn’t launch FinchTV, then try dragging the file onto the FinchTV icon, or launch FinchTV and then use the File Open menu option. If you have any trouble using FinchTV on your computer, remember that you’ll be working with your lab partners and so multiple students can analyze these chromatograms on one student’s computer. While this assignment could be completed with the pdf chromatograms, searching for sequences in those is more challenging, as is the visual analysis. The pdfs are included primary in case you’d like to have a beautiful, suitable-for-framing picture of your own DNA sequence!

page 4 of 9 Viewing DNA sequencing chromatograms NOTE: Copies of the 7 chromatogram files required for this activity can be found in the Canvas Files folder “sequencing files needed by all students” Open Plate2HERC-D7_PREMIX_Plate_Plate02_D07.ab1 (plate 2 well D7) in FinchTV. Use the menu option or icon above the sequence to select “Wrapped View” and then maximize the window on your computer screen. It should look like the image below. The height of the peak (more accurately, the area under the peak) indicates the quantity of DNA of that particular length. As you determined during a prior lab activity, the lower limit of detection is several million molecules of the same length. Most of the variation in peak size is irrelevant for us. More important is the Quality (Q) value of a peak. This is indicated graphically by the height of the small gray bar above the peak and more specifically by the Q value shown at the lower left corner of the window when a particular peak is highlighted. Q = –10 log 10 (P error ), where P is the probability of an error. Note the pattern here: P of 0.1 (10% probability of error) = Q10; P of 0.01 (1% probability of error) = Q20; P of 0.0001 (1 in 10,000 probability of error) = 40. A quality value of 20 or higher is considered the threshold for reasonable confidence in the data though we expect, and can have confidence in, a much lower Q value when it’s simply reflecting heterozygosity and thus a mix of two products of that length. Question 3: You’ll see that I’ve highlighted a base with a Q61 (as noted in the bottom left corner of the screen). What is the probability of that “G” being an incorrect base call?

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