Exercise: Without cover ... (Map Viewer)
You work with a patient who has had very patchy hair since birth. Hairloss can have a variety of causes and you will use bioinformatics tools to find out whetherthe herediatry hairloss of the patient can be related to anything in the human genom esequence.
- Laboratory protocol:
- Compare mRNA profile from patient with that from a person with normal hair growth.
- Isolate cDNA from patient and healthy person.
- Enrich cDNAs that are only present in healthy person.
- Sequence enriched cDNA.
- Bioinformatics protocol
- Highlight and copy the sequence.
- Open http://www.ncbi.nlm.nih.gov/.
- Find and click Map Viewer.
- Find and click Homo sapiens.
- Find and click BLAST search the human genome.
- Paste the sequence into the window, click Begin Search.
- Record request ID.
- Then, click Format!.
- Scroll down 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; what do you notice?.
- Scroll up and click Genome View.
- How many hits? What chromosome is/are the hit/s located on?
- Click the number underneath the chromosome.
- How many genes are shown as hits?
- What type of gene?
- Find the gene that contains eight matches with the query sequence.
- Zoom into this view (left mouse-click on vertical line, use combination of Recenter and Zoom. Zoom in approximately 32 times.
- Note the arrow next to the gene; what does it denote?
- Why doesn’t the cDNA align with the gDNA in one straight stretch?
- To determine the structure of the gene align the cDNA with the genomic DNA (gDNA).
- Go to http://pbil.univ-lyon1.fr/sim4.php/.
- Highlight and copy the cDNA sequence and enter it into the first window (cDNA).
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- Highlight and copy the genomic DNA (gDNA) sequence and enter it into the second window (Genomic DNA).
- 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 cDNA shorter than the gDNA?
- Which nucleotide stretch in the cDNA does NOT have an equivalent nucleotide stretch in the gDNA?
- What are the requirements of a sequence stretch that codes for a protein?
- How does the cDNA meet these requirements? Where in this cDNA is the 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 introns plus CDS?