Ders 10: Primer Tasarlama - Biyoinformatik-Lab

Primer3: http://biotools.umassmed.edu/bioapps/primer3_www.cgi
Primer-BLAST: http://www.ncbi.nlm.nih.gov/tools/primer-blast/
Exercises: A simple set of rules for primer sequence design is as follows

Primer Design Exercise

Currently Monsanto owns the patent on glyphosate, which is commonly known as Roundup®. It is the most popular herbicide used today because it kills a broad spectrum of weeds and is easily broken down into non-toxic compounds. The catch is that Monsanto also owns the patent on the gene that confers resistance to glyphosate, which they have transformed into several crops such as corn and soybean to make them “Round-up Ready”, or resistant to glyphosate. Many researchers are trying to find novel genes that will also confer resistance to glyphosate for both evolutionary and economic reasons. Recently, a Chinese group found a bacterium, Pseudomonas putida strain 4G-1, which is naturally resistant to glyphosate[1]. They have cloned the novel gene, aroA, that is significantly different in sequence from the previous AroA gene, and are hopeful that it will be another source of glyphosate resistance.

The AroA gene encodes the enzyme 3-phosphoshikimate 1-carboxyvinyltransferase, which plays a key role in the biosynthesis of aromatic amino acids. Glyphosate works by disrupting this enzyme and thus the biosynthesis of aromatic amino acids. Resistance is found by mutating the AroA gene such that glyphosate cannot bind to the resulting protein.

You have just been hired to select strains of Pseudomonas putida with the new aroA gene to provide a new glyphosate resistant cultivar. Your first challenge will be to create primer pairs (forward and reverse) that will amplify a portion of the aroA gene that contains the underlined region (see sequence below).

1. Given the sequence on the following page, choose forward and reverse primers that will amplify the underlined portion of the aroA gene.

        a. Underline the primer sequence on the following page. Then write out your primers below and indicate the 5’ and 3’ ends. Remember that the 3’ or reverse primer is the reverse complement of the template (think about which direction DNA extends).

         b. What is the size of your target DNA? (Note: each line contains 70 nucleotide bases)

>gi|51587624|emb|AJ812018.1| Pseudomonas putida aroA gene for 3-phosphoshikimate 1-carboxyvinyltransferase

5’-GATCATAAAACATGCTTGTATAAAGGATGCTGCCATGTTCCGTGAACTGGAAGCGAACAATCTTGCGGTA

TATCAGAAAAAGCCAAAGCTGATTGCAGTGCTTCTTCAGCGTAATGCTCAGTTAAAAGCGAAGGTTGTTC

AGGAGGATGAGTTCGAAAAGTCGGTAAGGCGTTTGTTGAACTTTGGTCATACATTGGGGCATGCCATCGA

AAATGAATATGCGTTGATGCATGGCCATGCGGTTGCTATAGGAATGACATACGCGTGTCATATTTCTGAG

CAATTGTCTGGATTCAAACAAACAAATCGCGTGGTAGAAGTGTTGGAACAATATGGGTTACCGACTTATA

TGGCATTCGATAGGGAAAAGGCTTTTAATCTGTTGAAAATGGACAAGAAGCGTGAAAAAAAGGAAATGAA

CTATGTGTTGCTGGAAAAAGTAGGGAAGGGAGTGGTGAAGAGTATTCCACTGGTTCAATTAGAAAAAATC

ATTCAAGCATTACCAAAGTGAAAGTAACAATACAGCCCGGAGATCTGACTGGAATTATCCAGTCACCCGC

TTCAAAAAGTTCGATGCAGCGAGCTTGTGCTGCTGCACTGGTTGCAAAAGGAATAAGTGAGATCATTAAT

CCCGGTCATAGCAATGATGATAAAGCTGCCAGGGATATTGTAAGCCGGCTTGGTGCCAGGCTTGAAGATC

AGCCTGATGGTTCTTTGCAGATAACAAGTGAAGGCGTAAAACCTGTCGCTCCTTTTATTGACTGCGGTGA

ATCTGGTTTAAGTATCCGGATGTTTACTCCGATTGTTGCGTTGAGTAAAGAAGAGGTGACGATCAAAGGA

TCTGGAAGCCTTGTTACAAGACCAATGGATTTCTTTGATGAAATTCTTCCGCATCTCGGTGTAAAAGTTA

AATCTAACCAGGGTAAATTGCCTCTCGTTATACAGGGGCCATTGAAACCAGCAGACGTTACGGTTGATGG

GTCCTTAAGCTCTCAGTTCCTTACAGGTTTGTTGCTTGCATATGCGGCCGCAGATGCAAGCGATGTTGCG

ATAAAAGTAACGAATCTCAAAAGCCGTCCGTATATCGATCTTACACTGGATGTGATGAAGCGGTTTGGTT

TGAAGACTCCCGAGAATCGAAACTATGAAGAGTTTTATTTCAAAGCCGGGAATGTATATGATGAAACGAA

AATGCAACGATACACCGTAGAAGGCGACTGGAGCGGTGGTGCTTTTTTACTGGTAGCGGGGGCTATTGCC

GGGCCGATCACGGTAAGAGGTTTGGATATAGCTTCGACGCAGGCTGATAAAGCGATCGTTCAGGCTTTGA

TGAGTGCGAACGCAGGTATTGCGATTGATGCAAAAGAGATCAAACTTCATCCTGCTGATCTCAATGCATT

TGAATTTGATGCTACTGATTGCCCGGATCTTTTTCCGCCATTGGTTGCTTTGGCGTCTTATTGCAAAGGA

GAAACAAAGATCAAAGGCGTAAGCAGGCTGGCGCATAAAGAAAGTGACAGAGGATTGACGCTGCAGGACG

AGTTCGGGAAAATGGGTGTTGAAATCCACCTTGAGGGAGATCTGATGCGCGTGATCGGAGGGAAAGGCGT

AAAAGGAGCTGAAGTTAGTTCAAGGCACGATCATCGCATTGCGATGGCTTGCGCGGTGGCTGCTTTAAAA

GCTGTGGGTGAAACAACCATCGAACATGCAGAAGCGGTGAATAAATCCTACCCGGATTTTTACAGCGATC

TTAAACAACTTGGCGGTGTTGTATCTTTAAACCATCAATTTAATTTCTCATGAATAGCTTCGGCCGCATC

TTCAGGGTGCATATTTTTGGCGAATCACATGGTGAATCAGTAGGCATCGTTATTGATGGTTGTCCTGCTG

GTCTGTCATTGTCCGAAGAAGATC-3’

2. For each primer you designed, use the website and the guidelines on the instruction sheet to determine whether they meet the basic primer requirements. Record the Tm, length, molecular weight, and possibility of secondary structures or primer dimers and use the statistics to qualify your decision to use or not use the primers for a PCR reaction.

3. To determine the specificity of your primer pair to the aroA sequence, run a nucleotide BLAST by following the directions on the instruction page.

a. What does the E-value indicate? What is another way to determine homology between two sequences?

           

b. Write down the organism and E-value score from the two highest matches

                 for each primer sequence. Did you get back the sequence you put in?

c. How many nucleotides aligned between your sequences and the first

                match for each?

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[1] Sun, Y. et al. 2005. Novel AroA with high tolerance to glyphosate, encoded by a gene of Pseudomonas putida 4G-1 isolated from an extremely polluted environment in China. Applied and Environmental Microbiology. 71 (8): 4771-4776