Exercise: Resistant to attack (BLAST)
Tuberculosis (TB) is an infectious disease caused by the bacterium Mycobacterium tuberculosis. In TB patients who do not respond to penicillin antibiotics any more M. tuberculosis has become resistant to this antibiotic type.
Research objective: Identify the genetic basis for this resistance.
Use this lab protocol to yield the resistance-causing gene(s):
- Grow bacteria in culture,
- challenge resistant strains with penicillin,
- isolate entire mRNA,
- reverse transcribe mRNA into cDNA,
- enrich cDNA by subtractive hybridization,
- clone resistance-specific cDNA,
- determine nucleotide sequence.
Bioinformatics protocol
To determine what causes the bacteria to be resistant to penicillin use the nucleotide sequence for a BLAST search.
- Copy sequence, open the NCBI Internet site at http://www.ncbi.nlm.nih.gov/.
- Click BLAST, then click Nucleotide-nucleotide BLAST (blastn).
- Paste sequence into window, click BLAST!.
- Record request ID.
- Then, click Format!.
- Record the Score and the E-value for the first hit.
- Which E-values denote higher significance for a hit? High? Low?
- What does e-06 denote?
- Find the hit with the gi number gi|1519647.
- Click on the Score for this search hit to view an alignment between the matching regions of the query with the hit. How many nucleotides match?
- Record the position numbers for the start of the alignment and the end into this worksheet.
- Then click the gi number in front of the search hit.
- On the next page identify the organism the DNA was derived from and the gene.
- To determine the structure of the gene align the cDNA with the gDNA:
- Highlight and copy the sequence in the Genbank entry, go to http://pbil.univ-lyon1.fr/sim4.php/ and enter it into the second window (Genomic Sequence).
- Into the first window (cDNA) enter the cDNA sequence above.
- Click SUBMIT.
- Click 2) Visualize the alignment ....
- Click Open. (If this doesn’t work you may have to download the viewer from ftp://pbil.univ-lyon1.fr/pub/mol_phylogeny/lalnview/.
- Why is the DNA shorter than the gDNA?
- Which nucleotide stretch in the cDNA does NOT have an equivalent nucleotide stretch in the gDNA?
- What is required of a nucleotide sequence to be able to code for a protein?
- How does the cDNA meet these requirements? Where in this cDNA is the sequence which codes for an amino acid sequence (coding sequence, CDS)?
- Use a tool denoting Open Reading Frames (ORFs) to identify the coding sequence in the cDNA.
- Why is the cDNA longer than the CDS?