Since my last post, I have been spending a great deal of time doing research on the genetic sequences of my target species and attempting to identify primer sequences that will be successful at DNA amplification across my chosen organism set. It is a bit of a painstaking process, as I am manually looking for nucleotide sequence similarities among the 16s ribosomal genes of Salmonella enterica, Klebsiella oxytoca, and Serratia marcescens instead of using a sequencing program. This means I take a piece of code that looks like this: (actual S. enterica 16s sequence)
cagagatggatttgtgccttcgggaactgtgagacaggtgctgcatggctgtcgtcagctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcgattaggtcgggaactcaaaggagactgccagtgataaactggaggaaggtggggatgacgtcaagtcatcatggcccttacgaccagggctacacacgtgctacaatggcgcatacagaagtcggaatcgctagtaatcgtggatcagaatgccacggtgaatacgttcccgggc
and compare it to something like this: (actual K. oxytoca)
atgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatattgcacaatgggcgcaagcctgatgcagccatgccgcgtgtatgaagaaggccttcgggttgtaaagtactttcagcggggaggaagggagtgaggttaataaccttattcattgacgttacccgcagaagaagcaccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttaatcggaattactgggcgtaaagcgcacgcaggcggtctgtcaagtcggatgtgaaatccccgggctcaacctgggaactgcattcgaaactggcaggctggagtcttgtagaggggggtagaattccaggtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggccccctggacaaagactgacgctcaggtgcgaaagcgtggggagcaaacaggat
and this: (actual S. marcescens)
gaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatattgcacaatgggcgcaagcctgatgcagccatgccgcgtgtgtgaagaaggccttcgggttgtaaagcactttcagcgaggaggaaggtggtgagcttaatacgttcatcaattgacgttactcgcagaagaagcaccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttaatcggaattactgggcgtaaagcgcacgcaggcggtttgttaagtcagatgtgaaatccccgggctcaacctgggaactgcatttgaaactggcaagctagagtctcgtagaggggggtagaattccaggtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcgggcccctggacgaagactgacgctcaggtgccaaagcgtggggagcaaacaggattagataccctggtagtccacgctgtaaacgatgtcgatttggaggttgtgcccttgaggcgtggcttccggagctaacgcgttaaatcgaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgatgcaacgcgaagaaccttacctactcttgacatccagagaactttccagagatggattggtgccttcgggaactctgagacaggtgctgcatggctgtcgtcagctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcggttcggccgggaactcaaaggagactgccagtgataaactggaggaaggtggggatgacgtcaagtcatcatggcccttacgagtagggctacacacgtgctacaatggcatatacaaagagaa
and attempt to find a sequence between 20-25 base pairs long that matches within each organism. It is not difficult, but it is certainly time consuming. Luckily, I love a good puzzle, this being no exception; therefore, I am glad to report that I am thoroughly enjoying the research portion of this project. I was also able to identify multiple sequence similarities, so I have placed an order for the appropriate primers and will be testing them for universality across all eight of my species as soon as the post arrives.
Whether or not my chosen target sequences will successfully amplify during the PCR process is an unknown at this point. Amplification should be successful with the three noted species, as the 16s gene of each strain carried in this lab has been mapped, and I used those maps (pictured above) to develop the primers. The remaining five species, however, are of particular strains that have not yet been mapped, and I cannot predict what the results will be for those strains. I will post the results when the experiment has concluded.
Until then, please enjoy the following Bio-Rad commercial. Cheers!
No comments:
Post a Comment