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In the next lab, you will analyze your PCR reaction products on an agarose gel that separates DNA molecules according to their sizes. 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. These records contain the CDS for your MET gene together with 1 kb of upstream and 1 kb of downstream sequence. In S. cerevisiae, these regulatory elements are usually located within 1 kb of the CDS.
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
Determining Annealing Temperatures for Polymerase Chain Reaction
Angela R. Porta, Edward Enners Determining Annealing Temperatures for Polymerase Chain Reaction. The American Biology Teacher 1 April 2012 74 (4): 256–260. doi: https://doi.org/10.1525/abt.2012.74.4.9
The polymerase chain reaction (PCR) is a common technique used in high school and undergraduate science teaching. Students often do not fully comprehend the underlying principles of the technique and how optimization of the protocol affects the outcome and analysis. In this molecular biology laboratory, students learn the steps of PCR with an emphasis on primer composition and annealing temperature, which they manipulate to test the effect on successful DNA amplification. Students design experiments to test their hypotheses, promoting a discovery-based approach to laboratory teaching and development of critical-thinking and reasoning skills.
The analysis of DNA by the polymerase chain reaction (PCR) is a remarkably simple technique that allows for amplification of minute quantities of DNA. The commercial availability of kits has made laboratories utilizing PCR more common in high school and undergraduate science classes. Students use PCR to determine DNA typing and fingerprints (Baker et al., 2002), to identify bacterial contaminants (Baker et al., 1999), and to clone for a particular gene of interest (Dong et al., 2008). Parameters for these experiments are often standard and preset. Students run the reactions without having a true appreciation for the critical experimental details required to amplify a specific segment of DNA.
The PCR cycle involves three steps: denaturation, primer annealing, and primer extension. Each of these steps requires incubation of the reaction mixture at different temperatures. In the first step, denaturation, the DNA is incubated at 93–95°C from 30 seconds to 2 minutes. This breaks the hydrogen bonds between the nucleotide base pairs (bp) and separates the two strands of DNA. In the second step, primer annealing, the reaction is incubated at 45–65°C for 45 seconds to 1 minute the presence of excess primers allows the complementary primers to hybridize to target DNA. The third step, primer extension, is conducted at 72°C from 15 seconds to 1 minute and involves DNA synthesis, in which the primers are used to synthesize two new daughter strands complementary to the original mother strands. Subsequent PCR cycles will replicate each PCR product in the reaction mixture, resulting in the exponential amplification of the DNA target sequence.
The early innovators of PCR needed to optimize this procedure. Initially, fresh DNA polymerase had to be added after each denaturation step. Eventually, a thermally stable form was discovered in the hot springs bacteria Thermus aquaticus (Taq), hence the term Taq DNA polymerase. Each incubation period required the transfer of test tubes by hand from one temperature to another until the advent of the thermal cycler, which regulates cycling temperatures automatically.
Even in the “real world” of scientific research, commercially available PCR kits are used, but two critical PCR components are usually provided by the scientist. Researchers supply their own primers, which are designed to anneal to a specific DNA sequence, and the DNA template to be amplified. An ideal PCR will be specific, generating one and only one amplification product, be efficient, yielding the theoretical two fold increase of product for each PCR cycle, and have fidelity, reproducing the exact sequence of the template. Each of these parameters is affected by variables within the PCR reaction mixture such as buffer components, cycling number, temperature, and duration of each cycling step, primer composition, and DNA template. In this laboratory exercise, students use two sets of primers to determine optimal annealing temperature on PCR product formation to optimize for efficiency of amplification. We use this exercise in a cell physiology laboratory course for upper-division undergraduates. It is also appropriate for AP Biology courses, where funding for more advanced laboratory exercises may be available.
The Appendix consists of three pairs of simulated chromosomes, represented as chromosome 1 (two versions), chromosome 2 (two versions), and chromosome 3 (two versions). Notice that each chromosome varies only in the number of VNTRs that are present. The VNTR is underlined a single time. The underlined pattern of nucleotides then repeats several times, and it varies on each version of the chromosome. For example, on chromosome 1, the VNTR pattern of ccttaacgat is present either 9 times or 21 times while the rest of the chromosome sequence is identical. Each student should be given two chromosome 1 sequences. It is important to note that in human populations, certain versions of a chromosome are more prominent then others. If you have 20 students, you should make 40 copies of chromosome 1, but you might have 30 of those copies containing 9 repeats of the VNTR while only 10 copies contain the 21 repeat version (as an example). In other words, just as students receive two copies of chromosome 1, one from their father and one from their mother, in this exercise students may have identical 9-repeat versions for both copies, some will have 9-repeat and 21-repeat versions, and some may draw both 21-repeat versions.
The second and third chromosomes are set up in the same fashion, although the VNTRs vary in length and sequence pattern. It is recommended that when making copies of chromosomes, you mimic real conditions by altering the ratio of each chromosome the students have access to. In the end, each student will have obtained two copies each of chromosomes 1, 2, and 3, each pair representing one chromosome from the student’s mother and one from the father. In this practice, we are limiting the experience to just three chromosomes with two VNTR variations per chromosome. In reality, VNTRs often exist in more than two variations per chromosome site and, thus, could be represented by more than two versions of each chromosome if the instructor so chooses. Also, the diversity in chromosome populations would only be dramatically increased when utilizing 23 pairs of chromosomes (the actual number of pairs that human cells contain) with multiple VNTRs possible per chromosome. Finally, the chromosomes provided in this exercise are displayed only in single-stranded format. Explain to each student that DNA is double stranded and that we must envision every G (guanine) interacting with a C (cytosine) and every A (adenine) interacting with a T (thymine). The chromosomes are not labeled with 5′ and 3′ ends. The relevance of this detail and its impact on primer design can be elaborated on if the instructor chooses to do so this requires a mere substitution of a 5′ label at each primed area designated with an asterisk. In an effort to prevent limitations in instructor use, the specifics of 5′–3′ directionality are not elaborated on in this exercise and are not required to conceptually grasp the general role of VNTRs in unique DNA pattern recognition.