Application of PCR and Bioinformatics
in the Identification of Unknown Bacteria
(Web-based Assignment)
Introduction:
Each student was assigned an unknown culture that contained a mixture of two different types of bacteria. The bacteria were isolated using traditional microbiological techniques, and two pure cultures were established. Students were then assigned sets of primers that could be used to identify their bacterial isolates, and were instructed in the application of the polymerase chain reaction (PCR). Following amplification, the nucleotide sequence unique to each isolate was inserted into a cloning vector and transferred to E. coli (transformation of E. coli with plasmid DNA). The transformed E. coli cells were plated on TSA-AMP and colonies were screened using gel electrophoresis to detect those carrying the target nucleotide sequences. When pure cultures of E. coli carrying the desired nucleotide sequences had been established, samples of the cloned DNA were extracted (Miniscreen – Rapid Extraction of Plasmid DNA), purified and sent to a sequencing laboratory. The sequences obtained are as recorded below.
Procedure:
1. Click on Safari to access the browser.
2. Access Geospiza and complete the green arrow BLAST tutorial as instructed.
2. Type the letters NCBI in the place of Google (upper right hand corner of your screen) and hit return.
3. Click on NCBI Home page and read the introduction provided.
4. Click on BLAST (upper central region of screen) and then scroll down to Nucleotide Blast.
5. Click on Standard nucleotide-nucleotide BLAST [blastn].
6. Highlight the nucleotide sequence you wish to use for your BLAST and copy it.
7. Paste the sequence into the horizontal box labeled search.
8. Hit the BLAST button, then on the next screen hit the Format button and wait for your results.
9. Scroll down to determine your results.
10. Look under sequences producing significant alignments, and click the blue area of highest score bit.
11. Record the required information about the organisms being identified.
12. Click on blue reference numbers to the right of Pub Med. then click on free full text article box.
13. Read the article and answer the questions.
Unknown Cultures as Assigned by Number:
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29
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30
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.
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27
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.
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28
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.
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References:
Nucleotide Sequences obtained from NCBI Gene Bank, National Library of Medicine (http://www.ncbi.nlm.nih.gov/)
Application of the Polymerase Chain Reaction (PCR) and Bioinformatics in Bacterial Identification - H. Wilson
Copyright 2003 Sierra College Biological Sciences Department
Last updated: 04/12/04.
