DNA sequence analyses fall 2023 (1)

pdf

School

University of Virginia *

*We aren’t endorsed by this school

Course

2010

Subject

Biology

Date

Feb 20, 2024

Type

pdf

Pages

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!

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version

Access to all documents
Unlimited textbook solutions
24/7 expert homework help

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?

page 5 of 9 Identifying single-nucleotide polymorphisms (SNPs) and relating genotype to phenotype Eye color We’ll start with the HERC2 SNP that influences eye color. Interestingly, this SNP is within an intron yet appears to influence the expression of a neighboring gene (OCA2). You may recall a recent example from class in which a sequence within an intron influenced gene expression… As indicated in a recent report in Human Genetics by Eiberg et al ., humans with brown eyes typically have one or both alleles including the HERC2 intron 86 sequence: TTCATTTGAGCATTAA A TGTCAAGTTCTGCACGCTAT while those with blue eyes have both alleles containing the sequence TTCATTTGAGCATTAA G TGTCAAGTTCTGCACGCTAT. In other words, these alleles show a simple dominant/recessive relationship with their influence on eye color. This SNP in the HERC2 intron 86 locus is known as rs12913832. Remember, this region is not within an exon of a protein-coding gene, thus there are no codons defined here. Let’s continue with Plate2HERC-D7_PREMIX_Plate_Plate02_D07.ab1 (plate 2 well D7) and determine this student’s genotype and expected phenotype. In the paragraph above, highlight and copy the bases before (or after) the A/G difference (i.e., a string of bases common to both alleles). Now, in FinchTV, paste that sequence into the Find Sequence box and hit return. Did you get a message “Pattern not found”? That may occur simply because the sequence noted above and the sequence currently shown in FinchTV are opposite strands of the DNA. Using the View menu, or icon above the sequence, select “Reverse Complement” and try your search again. Which base do you see at the SNP location (i.e., immediately preceding or following the common sequence that you searched for) for this student? You may also find it helpful to use the vertical and horizontal scale sliders in FinchTV to ensure your detection of heterozygosity, if present. Question 4: Is this student whose DNA was sequenced in plate 2 well D7 (filename Plate2HERC- D7_PREMIX_Plate_Plate02_D07.ab1) homozygous or heterozygous for this sequence? What color eyes would you expect this student to have? Question 5: Let’s now consider a different student, namely the student whose DNA was sequenced in plate 3 well C4 (Plate3HERC-C4_PREMIX_Plate_Plate03_C04.ab1). Is this student homozygous or heterozygous for this sequence? What color eyes would you expect this student to have? Question 6: Let’s consider yet another student , namely the student whose DNA was sequenced in plate 3 well H10 (Plate3HERC-H10_PREMIX_Plate_Plate03_H10.ab1). Is this student homozygous or heterozygous for this sequence? What color eyes would you expect this student to have? If you’re interested in learning more about HERC2 and these SNPs, see: http://genetics.thetech.org/node/607 http://tinyurl.com/nk98523 http://tinyurl.com/mqf5qfp or the report in Human Genetics by Eiberg et al . (posted on our Canvas site under Files)

page 6 of 9 Taste Let’s now consider the TAS2R38 target—which is a little more complex. There are 3 SNP in this gene influencing the ability to taste certain bitter compounds, including phenylthiocarbamide (PTC). There are two combinations of these three SNP that define the most common alleles. One SNP combination (PAV) results in codons specifying the amino acids P roline, A lanine and V aline at three locations in the polypeptide. The other common allele (AVI) results in A lanine, V aline, and I soleucine at these same positions. PAV is a taster and dominant allele (assume complete penetrance) while AVI is a non-taster and recessive allele. As illustrated below, the primers we used (known as rs1726866 and rs10246939) were designed to amplify a segment of DNA that includes only the second two of these three SNP. For our purposes here we’ll assume (recognizing that our assumptions could be wrong) that if we see AV, then the student is most likely PAV and if we see VI then the student is most likely AVI. The PAV (taster) allele includes the sequence below. Note the two SNP that we expect to detect, based on the primers we used for PCR, are underlined (the codon they are a part of is in bold). TCTGGTTTCTTCTGGGATGCTGACTGTCTCCCTGGGAAGGCACATGAGGACAATGAAGGTCTATACCA GAAACTCTCGTGACCCCAGCCTGGAGGCCCACATTAAAGCCCTCAAGTCTCTTGTCTCCTTTTTCTGC TTCTTTGTGATATCATCCTGT GCT GCCTTCATCTCTGTGCCCCTACTGATTCTGTGGCGCGACAAAAT AGGGGTGATGGTTTGTGTTGGGATAATGGCAGCTTGTCCCTCTGGGCATGCAGCC GTC CTGATCTCAG GCAATGCCAAGTTGAGGAGAGCTGTGATGACCATTCTGCTCTGGGCTCAGAGCAGCCTGAAGGTAAGA Having a single-stranded DNA sequence within a coding region, as above, but not knowing if the first base is the first, second, or third of a codon, nor whether it’s the coding or template strand, we need to consider six possible reading frames (three for each strand). Assuming no errors in the sequence data, and that the sequence is in fact within a coding region, it’s usually easy to identify the correct reading frame as the only “ o pen” r eading f rame (ORF)—the incorrect reading frames would show stop codons (noted with an “*”), just by chance. Entering the above sequence at https://toolkit.tuebingen.mpg.de/#/tools/sixframe shows only one completely open reading frame (lacking any * stop codons). That reading frame is indicated as “+2”. The + means that the DNA sequence we entered is the coding (versus template) strand and the 2 means that a codon starts with the second nucleotide (i.e., CTG is the first complete codon here). This DNA sequence encodes the following amino acid sequence (the AV of P AV shown below in bold): LVSSGMLTVSLGRHMRTMKVYTRNSRDPSLEAHIKALKSLVSFFCFFVISSC A AFISVPLLILW RDKIGVMVCVGIMAACPSGHAA V LISGNAKLRRAVMTILLWAQSSLKVR The AVI (non-taster) allele includes the following sequence (again, the two SNP are underlined and the codon they are a part of is in bold): TCTGGTTTCTTCTGGGATGCTGACTGTCTCCCTGGGAAGGCACATGAGGACAATGAAGGTCTATACCA GAAACTCTCGTGACCCCAGCCTGGAGGCCCACATTAAAGCCCTCAAGTCTCTTGTCTCCTTTTTCTGC TTCTTTGTGATATCATCCTGT GTT GCCTTCATCTCTGTGCCCCTACTGATTCTGTGGCGCGACAAAAT AGGGGTGATGGTTTGTGTTGGGATAATGGCAGCTTGTCCCTCTGGGCATGCAGCC ATC CTGATCTCAG GCAATGCCAAGTTGAGGAGAGCTGTGATGACCATTCTGCTCTGGGCTCAGAGCAGCCTGAAGGTAAGA The above sequence, in the correct reading frame, encodes the following amino acid sequence: LVSSGMLTVSLGRHMRTMKVYTRNSRDPSLEAHIKALKSLVSFFCFFVISSC V AFISVPLLILW RDKIGVMVCVGIMAACPSGHAA I LISGNAKLRRAVMTILLWAQSSLKVR Note that the sequences shown here are identical aside from those two nucleotides and amino acids. While PAV and AVI are the most common alleles, there are other known alleles. Less common alleles include AAI and PAI. Might you have one of these less common alleles?

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version

Access to all documents
Unlimited textbook solutions
24/7 expert homework help

page 7 of 9 If you’re interested in learning more about this gene and the SNPs, see the report in PLOS ONE by Robino et al . (posted on our Canvas site) or http://learn.genetics.utah.edu/content/inheritance/ptc/ PLEASE NOTE, the sequencing company made an error when naming these files—you’ll see that each of these filename references two different plate numbers. Be sure that you’re working with the indicated file in these next examples. If you’re finding the files in the Canvas folder that contains only seven different sequence files, and you focus on the well identifier, you can’t go wrong! Start with Plate9TASR-D1_PREMIX_Plate_Plate07_ D01 .ab1 Highlight about 20 bases before or after the base that differs between alleles as indicated on the previous page and then search for that sequence using FinchTV. Remember, a good scientist is thorough. It would be very wise of you to check both SNP locations in each file and not make assumptions. If you search for the first sequence after finding the later sequence, FinchTV may indicate “not found” simply because it searched only from the second location to the end of the sequence. Simply click search again to have it start from the beginning of the sequence and you should find what you’re looking for. Question 7: working with Plate9TASR-D1_PREMIX_Plate_Plate07_ D01 .ab1, is the student whose DNA was sequenced here homozygous or heterozygous for the TAS2R38 gene? Would you expect that student to be a taster or non-taster? Question 8: working with Plate7TASR-B12_PREMIX_Plate_Plate05_ B12 .ab1, is the student whose DNA was sequenced here homozygous or heterozygous for the TAS2R38 gene? Would you expect that student to be a taster or non-taster? Question 9: working with Plate7TASR-G6_PREMIX_Plate_Plate05_ G06 .ab1, is the student whose DNA was sequenced here homozygous or heterozygous for the TAS2R38 gene? Would you expect that student to be a taster or non-taster? Question 10: working with Plate7TASR-C8_PREMIX_Plate_Plate05_ C08 .ab1, is the student whose DNA was sequenced here homozygous or heterozygous for the TAS2R38 gene? Would you expect that student to be a taster or non-taster? Question 11: Do any of these four students appear to possess one of the less common A AI or P AI alleles of the TAS2R38 gene? If so, which? Question 12: While less common than PAV and AVI, ~1-5% of a population like ours is expected to have a PAI or AAI allele. How exactly do you suspect that the PAI and AAI alleles formed? Think deeply about these allele variations and what you’ve learned this semester. This is a higher-level analysis/application question. Finally , use FinchTV to analyze the chromatogram from your sequencing reaction. What do you discover about your genotype? Does your phenotype reflect your genotype as expected? Regardless of whether you selected the TAS2R38 gene or the HERC2 sequence for yourself, you are welcome to check your phenotype for PTC tasting at this lab. Just ask your TA for the tasting papers. Remember to get credit for your work! Be sure to complete and submit the associated Canvas DNA sequence assignment no later than 11:59pm Sun, Dec 3 rd . I urge you to complete this assignment during your scheduled lab period—if you choose not to, you risk forgetting to submit the assignment and you risk having no assistance for technical or other issues. Please take your time— every one of you should be able to earn all 35 points!

page 8 of 9 Primers for TAS2R38 (taste) 5’ TCTTGCCTAGGCCATGTATAGTGCTT 3’ 5’ TCTGGTTTCTTCTGGGATGCTGA 3’ > TAS2R38 (taste gene, 5’ to 3’) GAGGAAAGCCTTCCTGGCTGGGTTCCTGTGCAGGAATACAGACAATGCCAAATCTCTACAAATTTACCTTTCAGCTAGGTGA TTTTGGTAGGGTCTCCAACCAGAGAGACCAATACTTGGTACCAGGCAAAGCACCATCTTCAAAGGCAAATTGGGCATAGCCA GAAAGCCCACACAGGGCACCTACTGGCATCCAGGTCACCTACTGAGCAAATATCTGCTTCTCTTCCATCATTATAGAGTAGG TAAAAGTGCATTTCTGGATCAGTGTATTTTCAGACAAAACGACCCCTATCCTCTCGTGCCTCTTGAAGTTGAAGTTATGTGG TACCTCTTGAAGCTGGAATTATGACATAATTATCATTCCAAGTTTGTCTTTGTTTCCATGAAAATTGCGAAGTTTGAAGAGG ATTATATCCTTATTGTAGTAGTGAATATTTCCAAAAATGATGCTAGCACAAGTTCCGAGAAAGAGGTTTAACAGTCATGTTA AAAGCCAAACATATTATCTTGGGATTTAGATATTATTTCAGAAGAGATGATTTCTCATGGTTTGATTTATACCAATAATAAG CTATAGTATATAAAGGATTTACACCAAATACTTAATTATTTTGTTCATTTTATCAAAACAAACCTAGGGTACCAACATTATT CTTGCCTAGGCCATGTATAGTGCTTAATTATTTTATTCATCTATAAGTGAGTTCATTGATACAAAGTGGAGAGACTTTCAAT TGTGTTTTATAGAAGAAATTGGAAGGCTTTGTGAGGAATCAGAGTTGTATTCCTGAAGAATCAGAGGCATATTTATGAAGAC TCACAGGCGTATTAATGAAGAGCTCATTTCATGTCCATTCTCAGCACAGTGTCCGGGAATCTGCCTTGTGGTCGGCTCTTAC CTTCAGGCTGCTCTGAGCCCAGAGCAGAATGGTCATCACAGCTCTCCTCAACTTGGCATTGCCTGAGATCAGGATGGCTGCA TGCCCAGAGGGACAAGCTGCCATTATCCCAACACAAACCATCACCCCTATTTTGTCGCGCCACAGAATCAGTAGGGGCACAG AGATGAAGGCAGCACAGGATGATATCACAAAGAAGCAGAAAAAGGAGACAAGAGACTTGAGGGCTTTAATGTGGGCCTCCAG GCTGGGGTCACGAGAGTTTCTGGTATAGACCTTCATTGTCCTCATGTGCCTTCCCAGGGAGACAGTCAGCATCCCAGAAGAA ACCAGAAACAATAGGAAAGGAGGCACAGACCACAGATAGCAGAAGAGAAAGGAATAAAATAAATTGAGATCTTTAATCTGCC AGTTGAGCCTTGTATTGTTATTCATGAATAGCACAGTTGTGACTGTGAAGTGAGGTCTGCTAAAAAAGCACCAAACACAGAG GACAGTGCAGATGCAGGAGCAAAGAATAATACCCAGGAGCATCTGGGAGATCTTCCTGGAGACCCAGCTTGCCAAGCAGATC AGGAAGGTGTGAGAGAAACGGATGAGCTTGGAGCAGTAAAGCAGGCTGAGGCAGGCAGCAAGCCAGAGGTTGGCTTGGTTTG CAATCATCCATAGCATGATGATGGCTTGGTAGCTGTGGTTCAGTGGTTCACTCAACTTCTGGAAGTGGGTAAGCTGGATAGC ACTCAGGAACAGCAGTCCATGCAGGAAAAGCCGGCTGATGCTGAGACACAGCAGCACACAATCACTGTTGCTCAGTGCCTGC CTCTTCACTACATCCCAAAAATTCACCAAGAAAACGAAGGCATTGGTCAGAAACCCCACTGCAAACTCCAGGACTGAAATGA ACAGAAATGTACTCCTGACTTCATAGGACACAGTGCGGATGCGAGTTAGAGTCAACATGATGTCACTTCTCTAATTGGCTAT TCTACTTCTCTTCTCTAGTTGGCTAATCTAAAGACCTGGTTGCCACCCAGTGCAGAAAGGTAAATGTACGTTCCAAGCCAGG ACCTTTCCTTCACAAAGCATCCAGCTCTTTGCCTAGATCTATCCCAAAGCGAGCGCATAAAAAGTTCTGGCAGGAAACTTAG AGGGATGTTGTTTCTCTAGAACATCTCTTGGATACACGTAAGAGCTTATCATAGGGTGGAAGACTGATGGTTCTACCTCTTG GGTGATGCAAGATTAATAAATCCAGGTGTTCCTGTACAACCGCCTTCCCAGAAGGCCATAGCACAGCATCACGGATGCAAAC AAAACAGGGCCTCAATTTCCCCAATCCAGTTTTGCATGCCCATCTGGACCTGTGGGGGTGGCGAAGAGAACAGGATTTGCTT ATGGGTTTTGAGGCACCCTGCAGCAGGCAAAGCAGATATTCTTGGTGATCTAAGGTTTGAAATAGAGTCTCTTTTCATTTGC ATTCCTTCTCTCATCTGTGAAGTAATGACTCTTATCACAGACTGTGTTTTTAATATAGATAAATGCATGGTTCCTTTCCATG TGTCAGTGTATGATTGCAGTTTTATTTGCTGTTGCCAAAGGTTTCATGCAATCATCATTATTATGCCAGGCACTGTGCCTGG CAAGGAGAATATTAACATGAACAAGACTAGCACGCTGTCATGAAGGGAGAGGAAAGGGGGCATAGCTGGTTTGGATTATTAT TATGTTTATTTTTTAATTAGTATAAGTTCTGGGGTACATGTGCAGAACATGCAGGCTTGTTACATAGGCATACACCTGCCAT GGTGGTTCGCTGCACCCATCAACCCGTTATCTATATTAGGTATTTCTCCTAATGGTCTCCTTCCCCTTGCCCCCTACCCCCT GACAGGCCCTGGTGTTTGATGTTCCCCTCCCTGTGTCCATGTGTTCTCATTGTTCAACTGCCACTTATGAGTGAATTTCCCC CGATAAATCCTTTAAGAGTTATTGGTGCTGCCCCATTGGTGTTTTTCGATACTTTCTCTTTGTATTTTTTATGTTTTCTTTT GACTAAAGCTCCTTTTTTATTATTAGTAGTGACATAAGTCAGTGATTACTAAAGATGTTCACTTTTGTTTTATTCTGAACTG TTCATACGCAGATATTTATGTAGTCCTTATTTGTGCATCATGATGTAGCAGTTGTATTTAATTAATTGTGTTTGTGACCACT TCATTTGGGTCATTCATGCTTACAGAGTGTCAAACACAGAATTCTGCACTTTCAACTTAAGCTAATTTTCTCCTTGTCTTCA CCACACCAGGTGGCGGTGTTGCACTAAAGGTGTTTAGAAAAGATGTTTGCCTCCCTAAAAAGTCTAGGTAGAAACTTGCTCT ACTGCACAAGCAGAGATATATAACTATACAGTTAGAACCATTTATATCAAGATTATAATCTTTTATTAAAAAATAAAGTTCT TTATGTTATTTCTTGGAACACCAGATCCAATCTTAATTTTTCAATAATCAAGGGCTCTTTCAGAGAAAGGTAACACAAACCT TCATTGATACACCCCCTCCATTTGACTTATGTACACACAGTGTTTGGGATTATCTAAGTAATCTCCTTTGCAGATAGTCCTT AGCTATTGGCTATTTTTTGAAAAATGGTTTACTCAGGAAGCTAAAAACAGTAAAGTCAACTGCAATGCCAGTAAGAGCAGTG GTAGGAACACAAAAGCTCCTTATTTGTTATTAGTAGTGATATAAGTCAGTGACTACATCTTTATCAGATGTAATTAAAAAAA TCTTAGTCCCTGGATTTTGCAACTAAACAGATGTTAAAAATTCTTAACCTATATAAATTGGTGTTTCGACTTTTGCTGCCAT TTATTTTTGGAGATTGGAATTACATTCTTTGGATTGATTTTTAAAAATTGGTCTTTGTTAGCTTGACAGAAGAACACTATTA TTAGTAATATCAAACCTCATTCCCTTAAAATACCTTGCTTAGATGTTTCCAATGACTAGAGAAATGGACATACCAATATATG TTTATTAAGAGCAAAATATATTCCACCTGTTGTAATGGGCATTGAGGACACACTGGTAAACAAAG

page 9 of 9 Primers for HERC2 Intron 86 (eye color) 5’ GTTCATGTTCCCACCATCCT 3’ 5’ GCAAGTCAAGGTGCACTCAA 3’ > HERC2_Intron_86 (5’ to 3’) TAGTTGGAGCTTTCTAGAAGTTGGGTTAGTTTCTGGCAAGCAGCTTGACAAGATGATCTTGAAGGGGCTAATTACCATACGG TAGGGGCATCTCTGTATAGGGAAAAGTTATAGAATTGGATTGAGGACCACTAATCCTGATTGTGATTCTCTAATGTATTTTG TTGTCGAATATTATGGCCCATTCTTAGTGATTTAATAAAATTGCAATGTTTTGGAGTCCTGTCCCAGCAGGTTACTTTTTAA AGACCGGCTTTGTTTCCACTTGGCTAGAACGATTTCTGCTGAACAGGACTGGTACATTCATTTTACTTTCACGTGTGCCTGA GGGTGGAAGTGGTCAGGCTTGTGTTTAATGGAAAGACCATCTCTCTGATGAGAGAGGCAGAAGAAATCTTGGGCAGGTAAAA TGGATAGGAAACATAATTTCAATTACTGTTCAATAATAAAAAGTAGCAAACGATTAATTTTACCTTCTTTTTTTAATCCTTC AAAGTGGTGATGATAATAGGCAGCACAGAGGAAAAAAACCTTTCTTTGTAGGGAGAAAAAAAGTGAGAGGCAGGGCACGTCC CACCTGGCCCTGTGTGAGCGTTGTGCAGGCTGTGAGCGGGGCATGGGTGGGAGCTGGTGTGTGGTAGGGGAGCGGTGGGCAG CAGCATCTGGCACCTGCTGGGGCTGCAGGAGAGCAGGGGAGTGCTGAGGGCGTCTGAAAGGTACCTCTGGTCAGGGAATGTG TAGGCAGCCTCGGTTCTGGTCCTGGTTATGGTGAGGATCGGCCTCAGAGAGTGGGTGTTACTACTCCCGGTCCAGAGCACCA AAATGGACTCTCCAGGAAGGTGGGCGGGCTGGTGACTGGGAGGAGAGGGGAGTGCTCCTGTGGAGGGGGAGCCGCCCTGAGG TCCAGGCAGGGCCTCTGGGAGCAGGATGAGTCTGGGCTGTGCGAGAGGCATGGGGCTGGGGTGCCGCCAGGATACCCTTTCT GGGTCTCCTCTGGCGCTTGGGGCCCTCACCTCCTGCCTTCAGGGCTTGGAGTTCTTAGGCTTTGAGCTGGGCAAGGGCTCAG CCTCCACTGAGGGACCCTTGTGCTCCCACAGCCCACCCCTAACCGAAAGAAGGCACGATTGCCTCCTTGGCCAAGAGAGCTG AGACCCCTGCTGACCTGTCTCATCCCTTTCCATTGGCACGGCTGCGTGCCGCATCCTCTGCCCTTTCCCCACGGGCATGGCC GACCTGCTCCCTCCTGGGGAACGGCTGGGTCCTCACCGTGGTGCTGATGTTTGGCCACGCCCTGCTGTCTTCACCCCAGGCA CTCCCGATTTCTTTTAGTGTGATCCCAGCCAGGACCTTGTTTCCCCTCCCCGGTCTGTCTTTTCTGTCCCGATCCAAGGTCT GGCTGGCTGCACGGTGCTCCCTGGCCCCGTGGCTGCTGTATGGCAGGCAGTGGCGATCCAAGGTCCGGCTGGCTCCGCGGTG CTCCCTGGCCCGTGGCTGCTGTATGGCAGGCAGTGGCGATCCAAGGTCCGGCTGGCTCCGCGGTGCTCCCTGGCCCGTGGCT GCTGTATGGCAGGCAGTGGCGATCCAAGGTCCGGCTGGCTCCGCGGTGCTCCCTGGCCCGTGGCTGCTGTATGGCAGGCAGT GGTTTAACAGCAATCCCTGCCACGGGTGGGCTTGCTTGGCAGGGAAACCTTGACTTCAAGACCTGAGTCAGAGCAGGTGCTT GCCAGGCCCAGCCCTCCCTGTTCTGTGCCCTGTGTGTGCTGGGCTCTTCCAGCCTCCGGAACGCTGCTGGGTGGAGGTAGTT TCCTGAGGGACCTGCCTCTTGCCCGGCCATTGGCACTACCTGCCTGGCCGTCTGTCCCTGTGTGCTTCGGGGCCAGCCTGTT CCCCTGCACCCTCACAACTTGAGAAAAGGGAACTGGCAGTTTTCTGTCCTTAAGAAGGTCTTCAACACAGATTTTAAACAAA ACTATGTGATGATTTCTTCAGGAGTGATGCTTTCCATATCAGATTCTAAATTTTGTCTGTTTGATGTGTTTTAGGAGGCGGC TTTCCGGAAAGTAGTACAAGCAACTATGGTACGCGATCGTCAGCATGGCCCCGTCGTGGAGCTGAACCGCATCCAGGTAGCA CATGGAGATTACTCTCCAAGTCTGACAGCCTTAGAAGTGATGCTTTCGTGGGTACCTGGGCTGGGACGAGAGCGCTGGTAGC CTGCCATCCTGTGCACCCCAACTTTAAAGAGCAGGTGCCACCTTCCTTTTTGTGGGCTTCCTGTATGTGATGTGCTGGGGCT TCCAGGAATGTCAGTGTGTTTCTTTATACAGAAGTAATGAAGATTTATGTCAGATAGTCCAAAAGCACAAACACAGGTCAGC AAGAATGGGGAAAATAAAATCAGCCCTCTTCCCACTGCATGATGATAACCGCTGCTGTTACATTGGGAGGTGTGTGTCTATT TGTTACAGCCAAAGCAGGATCACTGCCTTTTGAAATTTGCTGCTCTCCTCCCTGCCATGGAATAGGGTCCTGTGGGGCTGCT TGTGTTTGGCAGCTGCCTGCAGCCTGCTCCTCTCTTGCAGGGGTGTGTCCTGGTTTACTCTGTCAGATTTCTCTGATGGTGC TGGGGGGCCGTGCCCACTTTTCTGTGGTACAGGCAGGGCTATCTGGGTGTATCTTCGGTCACCCCTTTTGTTAAGTTCTGAG AAGTGGAGTGGGTCAGAGGCCCAGTGACAATTTGATGATACATCCGATTATAGGTTATAACAGTTCATGTTCCCACCATCCT CACACCCTCCCCACCACTGGTAGTTTTCTTTGCCAATTTCATAGGCCAGAATTACTAATACTGTCTCATGGGTAGTAATCAA AGAAACGACAAGTAGACCATTTCTAAATGTATACTGCTTCAAGTGTATATAAACTCACAGTTAAAAACAATTAATTAAAAAC AAAGAGAAGCCTCGGCCCCTGATGATGATAGCGTGCAGAACTTGACATTTAATGCTCAAATGAAACTGGCCTCGCCTCTTGG ATCAGACACAGAGAGCCATGAAGAACAAATTCTTTGTTCCTGTACCTTTGTATTAACACATGATTTTTACCCTGATGTTGAT TACAAGACTAGAAATGTTTTCAGAGTTATACTTGGGGGCATTTTGAATTAAGACACGAAACTCTTGTCCTTTGTTGGACAGC TCATGTGACCACATTGATGGAGCTGGTCTCTTCACTGACGTCAGAGTGTTTAATGCTAAGCTGTACGTCAGTCACGTCCTCA CATAAATAGGTTGTTTAACTAGGTTCATAAAAAAGCTTTTATTTCCCTACCTTGGACGATGGCTTCAGTTTGCTGTGCATCA TAAACATTATAGCTTGTAGGCAAGGCCAGCTGATGGCTTCTGCGTTGTCTAACCCCCGCGATTGAGTGCACCTTGACTTGCA CCTCCCTCAAAACCGCACGCAGACTTTTTGGGCCAGGGGCTCTCAGGGTGGGTATTCAAGCCTGGGGGACTCTGGGAATCAG TGAAGTTTTGATAAACTAGGATGCAGGTTAGGCACCCTTGTTTCTGTCTTTGTATCATCTGAAATTTTTAAGAAGTCATTAC ATGCTGAGCACCTCTTTCTTACTCATCTTGCTATCCCCAGCTTGTGTTACAAAAACGTTCAGTATGCAATTGAAAAAACAAA CTGGGTAAATGGCAAACAAATTTATATTAGTCGTTGGTGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCTGACGTG GATGGATTGCTTGAGCCTAGGAGTCTGAGACCATCCTGGGCAACGTGATAAAACCCCATCTCTACAAAAAATACAAAAATTA GCCTTGTGTGGTGGTGGGTGCCTGTAGTCCCAGCTACTTGGGAGGCTGAGGCAGGAGGATTGCTTGAGCCCGGGAGGCAGAG GTTGTAGTGAGCCGAGATCATACCAGCGTACTCCAGTCTGGGCGACAGAGCCAGACACTGTCTC

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version

Access to all documents
Unlimited textbook solutions
24/7 expert homework help

DNA sequence analyses fall 2023 (1)

Related Documents