CSB349H1 Lecture Notes - Lecture 3: Gene Duplication, Crispr, Transcriptomics Technologies
14 May 2018
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*Control (green): red = enrichment
yellow = no enrichment
*if outside the red lines; protein is
found, if between not found
Lecture 3(b): Transcription
Complications with Transcriptomic Analyses:
• Genes are created from other genes, by gene duplication; genes that have been recently
‘created’ will have highly similar sequences
o mRNAs from gene families with similar sequences are difficult to quantify
• Different mRNAs from the same gene (due to alternative splicing) are difficult to distinguish
o Transcripts from more than one gene may hybridize to the same oligos on the array
§ This is because gene is sliced up into small fragments; those regions may arise
from one exon; unable to identify which alternative transcript it came from
• Specific oligos must be available on the array to distinguish them
ð Transcriptomics only tell us the change in abundance of each mRNA
o Does not tell us whether the rate of transcription or transcript degredation has changed
o Does not tell us whether protein levels have changed
Genome Annotation:
• Ability to discover new transcribed units in the genome
o Sequencing transcripts within the genome is not all coded by exons or protein coding
regions of the genome
§ There is a lot of the genome that is not associated with known genes
§ We can discover that there is high transcription of mRNAs where no exons
are found within the genome which allowed for the discovery of new genes
Protein-DNA Interactions:
• Major factor that controls transcriptions are proteins (i.e. transcription factors)
o Genomic technologies: ChIP-seq (or ChIP-chip) combines chromatin IP with
microarray or sequencing to identify protein DNA interactions that occur in live cells
§ ChIP-chip => hybridization | ChIP-seq => sequencing
• Key Idea:
o Require an antibody for our protein of interest
ChIP-seq & ChIP-chip:
a) Chromatin is cross-linked via molecular glue
o Will glue any protein-DNA interactions
b) Anti-body is added to the chromatin and is allowed to bind to
the protein of interest
o Immunoprecipitate regions where antibody is found
c) Obtain small fragments of protein-DNA attached to anti-body
o Reverse the cross-link and isolate DNA sequence; which
can be either sequenced or hybridized via microarray
ð High quality anti-body are optimal: high avidity and specificity for binding
ð Amplify the ChIP enriched DNA using LM-PCR (ligation mediated PCR)
Document Summary
Lecture 3(b): transcription: genes are created from other genes, by gene duplication; genes that have been recently. This is because gene is sliced up into small fragments; those regions may arise from one exon; unable to identify which alternative transcript it came from: specific oligos must be available on the array to distinguish them. Transcriptomics only tell us the change in abundance of each mrna: does not tell us whether the rate of transcription or transcript degredation has changed, does not tell us whether protein levels have changed. Genome annotation: ability to discover new transcribed units in the genome, sequencing transcripts within the genome is not all coded by exons or protein coding regions of the genome. There is a lot of the genome that is not associated with known genes. We can discover that there is high transcription of mrnas where no exons are found within the genome which allowed for the discovery of new genes.
