C14 Genomes and Genomics
25 important questions on C14 Genomes and Genomics
Pyrosequencing
- How are the DNA strands in step 1 obtained?
- why is there a PCR reaction in step 2?
- why is each bead placed in a single well in step 3?
- This is e.g. Genomic DNA (to make sure the whole genome is sufficiently represented a high coverage is needed), or exomic (only exons, for protein encoding genes) ....
- to amplify the fragment so that a stronger signal will be obtained in the following reactions
- you want to study individual beads for whether a base is incorporated or not
Why do not all wells show light in every round?
Where does gene transcription start and stops in this figure?
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How many 5' and 3' splice sites are there in this figure?
WHy are there no codons in the intron?
What is the 3' UTR between the translation termination site and the polyadenylation site?
Function of UTRs is not yet clear but may have regulatory functions and affect stability of the mRNA
What is the 5' UTR and why is it located behind the promoter and before the translation initiation site?
Indicate which binding site is DNA and which RNA
Why are the ribosome binding site and ORF drawn on the non-template strand of genomic DNA?
What is synteny? Which chromosome had diverged more since the last common ancestor, the human chromosome 17 or the mouse chromosome 11?
Why fo mainly exonic fragments bind to the immobilised probes?
Why would you sequence only the exome if you want to study the causal mutation of a patient?
- What is the control in step 2?
- why is the intensity of the green colour lower in some wells than in others in step 5?
- why are some wells green and others red in step 6?
- E.g. Cells that have been grown under normal rather than 'experimental' conditions
- because less mRNA was present, lower expression --> less cDNA
- some have higher (red) expression than control, others lower (green)
- How can you select a yeast cell that carries both plasmids?
- why is there only expression of the lacZ reporter when the two proteins interact in the cell?
- By transforming the plasmids to yeast and selecting for transform ants that are both Trp+ and Leu+ (can grow in the absence of tryptophan and leucine)
- when both proteins are present in the cell and interact, the Gal4 binding domein and Gal4 activation domein will be together and form an active transcription factor for the expression of the LacZ gene.
In the ChIP procedure, why is the protein cross-linked to the DNA in step 1?
Why does the antibody only bind to the orange protein in step 3?
Why is DNA amplified and sequenced at step 4?
What is the light-green 'mutant' segment in gene A?
Why are two crossovers required for gene replacement?
What happend to the plasmid DNA (indicated in red) in the lower part?
What would you do after isolating a mutant with the disrupted gene A?
Why is this an example of 'reverse genetics'
What dsRNA sequence is synthesised in vitro (left part of the figure)
What does it mean that the arrows point in different directions (middle part of the figure)?
What is the template for the transcription of the two RNA molecules?
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