In the next lab, you will analyze your PCR reaction products on an agarose gel that separates DNA molecules according to their sizes. To interpret those results, it will be important to have calculated the expected sizes of the PCR products from your reactions. To assist those calculations, prepare a simple map of the genomic region containing your gene with the primer sequences aligned against the genome sequence. All PCR products should contain a portion of the MET gene’s 5’-flanking region because of primer A. A PCR product may or may not contain portions of the MET gene’s CDS, depending on whether you are analyzing a strain with the native or disrupted MET gene. To constsruct your map, you can take advantage of special genome sequence records prepared by SGD curators. These records contain the CDS for your MET gene together with 1 kb of upstream and 1 kb of downstream sequence. SGD curators generated these records because researchers are often interested in studying regulatory elements that control transcription of a gene and the processing of gene transcripts. In S. cerevisiae, these regulatory elements are usually located within 1 kb of the CDS.
The figure on the right shows a map for the SAM1 gene. The binding site for primer A is 357 nucleotides upstream of the SAM1initiation codon (nucleotide 1001). Primer B-anchored products add 280 bp of CDS to the PCR product. The expected size of the PCR product is 357 + 280 bp, or 637 bp. If the deletion strain had been used for PCR, theSAM1 primers A and B would not generate a PCR product. Instead, SAM1 primer A andKANR primer B would generate a 607 bp (357 + 250) product, because the KANR primer B binds to nucleotides 231-250 of the KANR CDS.
You will need two browser windows for this exercise. Each member of the group should work with a single gene.
Find the genomic sequence for your gene.
• Navigate to your gene’s summary page in the SGD (yeastgenome.org)
• Click the Sequence tab at the top of the summary page.
• Cursor down to the gene sequence for S288C. Select “Genomic sequence +/- 1kb” from the dropdown box.
• Note below the starting and ending coordinates for the sequence and calculate the length of the sequence. (You should see the ATG start codon at nucleotides 1001-1003.)
Length of sequence (bp) __________
Length of the coding sequence ___________
Align the primer sequences with the genomic sequence.
To find the position on the primers in the genomic sequence, we will use NCBI’s BLAST tool. BLAST stands for Basic Local Alignment Search Tool and can be used to align either protein or nucleic acid sequences. You will learn more about the BLAST algorithms in Chapter 9.
Direct your browser to the NCBI site and select BLAST from the list of resources on the right.
Select Nucleotide BLAST from the list of Basic BLAST programs.
Click the box “Align two or more sequences.” Copy the “genomic sequence +/- 1kb” from
SGD and paste the sequence into the lower Subject Sequence box.
Type the Primer A sequence for your gene in query box.
Adjust the BLAST algorithm for a short sequence. The primer sequences that we are using
are 25 nucleotides long. This is shorter than the default value of 28 for “words” in BLASTN (the algorithm for comparing nucleotide sequences). BLAST will not align two sequences if the match is smaller than 28 nucleotides. Expand the “algorithm parameters” at the bottom of the page. Select a “word size” less than 28.
Click BLAST. The BLAST results bring up a table that shows each match between your primer and the genome sequence. The top result should be a perfect match between your primer and the genome sequence. (Check your typing if it isn’t a perfect match!) Record the starting and ending nucleotides in the genomic DNA sequence where it matches the primer sequence.
Repeat the BLAST alignment for primer B. Click “Edit and Resubmit” at the top of the BLAST results page. Clear the query box and type in the sequence of primer B. Click BLAST and record the alignment results. In the results, note that the primer nucleotide numbers are ascending, while the genomic DNA nucleotide numbers are in descending order. This is because Primer B sequence is the reverse complement of the gene sequence.
Draw a map of your gene and primer binding sites in the space below. Include the start codon and distances in bp.
Calculate the sizes of the PCR products that would be generated with:
Primer A and Primer B
Primer A and KANR primer B