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
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?
[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
