Updated August 2016 These instructions are modified from those in pages 12-16 of Using a Single Nucleotide Polymorphism to Detect.... The NCBI website is a constantly changing database, every day new entries are made as the field progresses. Therefore, some of the instructions were modified to reflect changes since this protocol was created in 2006. READ and follow the directions and answer the questions in italics. I. Use NCBI BLAST to Find DNA Sequences in Databases (Electronic PCR) The following primer set is used in the Polymerase Chain Reaction (PCR) to isolate a portion of the PTC tasting gene: 5 -CCTTCGTTTTCTTGGTGAATTTTTGGGATGTAGTGAAGAGGCGG-3 (Forward Primer) 5'-AGGTTGGCTTGGTTTGCAATCATC-3' (Reverse Primer) ***NOTE: You should first copy the primer sequences into a text file. This way you can save the sequences for easy copy and paste throughout the protocol. Question 1. What is the role of a primer in polymerase chain reaction (PCR)? 1. Initiate a BLAST search. a. Open the Internet site of the National Center for Biotechnology Information (NCBI) www.ncbi.nlm.nih.gov. b. Click on BLAST in the column to the right under Popular Resources.
c. Click on the link nucleotide BLAST under Basic BLAST. d. Enter the primer sequences (only the letters) into the text area underneath Enter Query Sequence. f. Under Choose Search Set, select the Nucleotide collection (nr/nt) database from the drop-down menu. g. Under Program Selection, select somewhat similar sequences (blastn). h. Click on BLAST. The query sequences are sent to a server at the National Center for Biotechnology Information. The BLAST algorithm matches the primer sequences to the millions of DNA sequences stored in its database. A page with the status of your search will be displayed until results are available. This may take only a few seconds, or more than a minute if many searches are queued at the server.
2. The results of the BLAST search are displayed in three ways as you scroll down the page: a. First there is the Graphic Summary. This graphical overview illustrates how significant matches, or hits, align with the query sequence. Matches of differing lengths are coded by color.
b. Followed by Descriptions, a list of significant alignments, or hits, with links to Accession information. c. Next Alignments displays a detailed view of each primer sequence (query) aligned to the nucleotide sequence of the search hit (subject). Notice that a match to the forward primer (Range 1), and a match to the reverse primer (Range 2) are within the same Accession. Question 2: Do you notice position 43 of the forward primer is missing? What does this mean? 3. Analyze highly aligned sequences. a. Under Descriptions, notice the E-value column toward the right. The Expectation or E-value is the number of alignments with the query sequence that would be expected to occur by chance in the database. The smaller the E-value is, the higher the probability that the hit is related to the query sequence. Question 3: What is the significance of an E-value of 2e-11?
b. Look at the names of significant alignments with E-values less than 0.1. Question 4: What do the names have in common? Question 5: Does this make sense? c. Scroll down to the Alignments section to see where the two primers align in the subject sequence of a hit. d. The lowest and highest nucleotide positions the subject sequence indicate the borders of the amplified sequence. Subtracting the lowest from the highest nucleotide positions in the subject lines gives the difference between the two coordinates. Adding 1 nucleotide (representing the missing query nucleotide) to the result can be used to calculate the length of the PCR product amplified by the two primers. ***NOTE: This is also called an amplicon. II. Find and Copy the Human (Homo sapiens) PTC Taster and Non-taster Alleles 1. In the list of significant alignments, select the hit containing the human taster allele with the Accession Number AY258597. 2. Click on the Accession Number link at right to open the sequence datasheet for this hit. 3. There is basic information about the sequence at the top of the report, including the number of basepairs, database accession number, source, and references.
4. In the middle section of the report called Features, note the gene and regulatory features, with their beginning and ending nucleotide positions (#,, #). Question 6: Identify the feature(s) contained between the nucleotide positions identified by the primers. 5. The bottom section of the report lists the entire nucleotide sequence of the gene or DNA sequence that contains the PCR product. 6. Return to the top of the page and click on FASTA below the GenBank Accession number. 7. Highlight all the nucleotides in the sequence.
8. Paste the sequence into the Sequence Massager program and delete non-nucleotide characters and spaces. http://www.attotron.com/cybertory/analysis/seqmassager.htm 9. Copy and paste the massaged sequence into a text file or word document. 10. Repeat Steps 1 9 to find the human non-taster allele (Use Accession AY258598). IV. Compare the human PTC taster allele to the human PTC non-taster allele. 1. Use the ClustalW alignment program at www.bioservers.org from Cold Spring Harbor Laboratory. a. Click ENTER under Sequence Server.
b. Click on CREATE SEQUENCE at the top of the page. A new window will open for you to add sequences to the workspace. c. Copy the human taster allele, and paste it into the Sequence window. Enter a name for the sequence, and click OK. Your new sequence will appear in the workspace at the bottom half of the page. d. Repeat this for the non-taster allele, forward primer, and the reverse primer sequences. 2. Select the PTC taster and PTC non-taster alleles. Click on Compare in the grey bar. The default operation is a multiple sequence alignment, using the CLUSTAL W algorithm, which will attempt to align each nucleotide position.
a. The results will appear in a new window. This may take only a few seconds, or more than a minute if a lot of other searches are queued at the server. The sequences are displayed in rows of 25 nucleotides. Yellow highlighting denotes mismatches between sequences or regions where only one sequence begins or ends before another. b. To view the entire gene, enter 1100 as the number of nucleotides to display per page, then click Redraw. Question 7: List the nucleotide position(s) and nucleotide differences of any additional SNP(s). Question 8: Count triplets of nucleotides from the initial ATG start codon to determine codon(s) affected by SNP(s). 3. Find the binding sites on those sequences for the primers by aligning each primer sequence to allele sequences. Compare forward primer vs. human PTC taster vs. human PTC non-taster as just completed in the Sequence Server between the two allleles. Write down position of where the forward primer aligns to the alleles. Then compare the reverse primer to the alleles and write down the position of where the reverse primer aligns to the alleles. The sequence area from the beginning of the forward primer to the end of the (reverse complement) reverse primer is the amplicon or amplified product. ***NOTE: DNA is double stranded. This is only one strand, therefore you will not be able to find the reverse primer, as it is on the complementary strand. You need to look for the reverse complement of the reverse primer. Use the Sequence Massager to convert the reverse primer to the reverse complement. In the PCR process, the forward primer binds to the complementary strand 3-5 of DNA and its sequence almost always matches the sequence on the template strand 5-3, being the latter what can be found in NCBI databases. However, the reverse primer must bind to that template strand, so it s
actual sequence is complementary and antiparallel to the sequence obtained from the database. In order to match the alignment, you need to convert every nucleotide of the reverse primer sequence to its complementary, as well as invert the order of the nucleotides. Once you have the reverse complementary sequence to the one given, you can perform the alignment. ***NOTE: There is a mismatch between the forward primer and the alleles that end in GGCAG not GGCGG. This nucleotide change creates a sequence GGCC, which corresponds to the restriction site for HaeIII restriction enzyme that we will be using in the digestion step of the experiment. The amplicon will contain that nucleotide change! Question 9: What does the initial stretch of yellow highlighted sequences mean? Question 10: Where do the primers bind to the sequence? Question 11: What is the position of the amplicon within the two human alleles? 4. Calculate the size in basepairs (bp) of the specific sequence or amplicon that will be obtained by PCR. The first position of the alignment between the forward primer and the alleles is the beginning of the amplicon. The last position of the alignment between the reverse primer and the allele sequence is the end of the amplicon. If we calculate (final position start position + 1) we will have the length in bp for the fragment of the sequence that will be amplified by the PCR. Question 12: What is the size of the amplicon in basepairs? Question 13: At what position in the gene is the SNP examined in the experiment, and what is the difference between taster and non-taster alleles within the amplicon?
V. Translate the full nucleotide sequences of the taster and non-taster alleles to amino acid sequences. 1. Use the Translator tool from the Expasy server http://web.expasy.org/translate/ 2. Paste one of the allele DNA sequences into the text box. 3. Click on TRANSLATE SEQUENCE ***NOTE: The results window shows 6 translations for the 6 potential reading frames. The allele sequences are complete coding DNA sequences (cds) and only one of the possible reading frames represents the entire protein sequence from beginning to end. 4. Click on the first Methionine
5. Select the blue title of the reading frame with the entire protein sequence that is open, or all highlighted in red. 6. Click on the link to FASTA format. 7. Copy the FASTA format of the sequence and paste in your word document. Above the sequence, create a label for either the taster or non-taster allele.
8. Repeat translation steps for the second allele. VI. Compare taster and non-taster amino acid sequences. 1. Use the T-Coffee aligner from the EBI server http://www.ebi.ac.uk/tools/msa/tcoffee/ (*NOTE: For a better visualization of results, the Java applet Jalview is included in the results summary tab. However, if you are a Chrome user, you will find that Chrome does not support Java, for some really strange unknown reason. Try with Firefox instead and play with the visualization options that Jalview offers to highlight specifically the mismatches between both amino acid sequences.) 2. Paste your sequences into the sequence input area. Label your sequences with >taster and >nontaster above each sequence as shown below. The > means that it is the name of the sequence. 3. Click on Submit to align the translated sequences.
4. Click on the Result Summary tab. 5. Under Jalview, click on Start Jalview. You may need to allow the use of the Java applet. A new window will open with the aligned sequences.
6. Click on the Format tab and click on Show nonconserved to check this off. All other options should stay in the default position. Check that the parameters are the same as those above. Question 14: What are the amino acid changes? Question 15: What type of mutation are these changes?