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17 Cards in this Set
- Front
- Back
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What is Shotgun Sequencing? |
A cost effective method of sequencing DNA. Small fragments of DNA is sequenced at random, and DNA fragments (reads) are then re-assembled to form the target sequence. |
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What is Illumina Sequencing? Describe it's protocol. |
Technique used to determine the sequence of multiple DNA strands at the same time. 1. Randomly fragment DNA, and ligate adaptors onto both ends. 2. Immobilize the DNA fragments onto an array with primers matching the adaptors. 3. Denature DNA into single-strands, and amplify using primers already present on the array (bridge amplification). 4. Repeat process multiple times to generate numerous ssDNA. 5. Place mix of random primers, and DNAP onto the array along with fluorescently labelled nucleotides. 6. As the nucleotides associate with the ssDNA, sequence can be determined by the colour of fluorescence. |
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What is the transcriptome? |
The collection of all RNA transcripts in a cell at any given time. |
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What is the goal of transcriptomics? |
1. Identify which mRNA's are present 2. Quantify how much of each transcript occurs |
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What is RNA-seq? |
Most direct method to analyze the transcriptome. 1. Using reverse transcriptase convert mRNA into cDNA. 2. Sequence resulting cDNA. |
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What are alternative methods of transcriptome analysis? |
Can use hybridization - short oligos are immobilized and cDNA is labelled and hybridized to oligos. (Chip microarray) |
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What is the advantage of sequencing over hybridization? |
Sequencing can in theory identify transcripts that are not known in advance. |
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How do you compare two transcriptomes using microarrays? Explain. |
1. Label the two transcriptome sample with different fluorescent dye. 2. Reverse transcribe mRNA into cDNA for both samples. 3. Mix samples and hybridize onto the same array. 4. Determine relative gene expression of both samples from the colour and degree of fluorescence. |
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What is cluster analysis? |
A method to determine patterns in gene expression of two different transcriptomes, and group similar genes together. Genes with similar expression patterns often have similar functions. |
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What are some challenges in transcriptome analysis? |
mRNA from similar gene families are difficult to quantify and separate. -Transcripts from more than one gene may hybridize to the same oligo - Sequencing reads from more than one gene may align to the same genomic region -> difficult to discern alternative splice forms |
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What is ChIP-Seq? Explain it's mechanism. |
ChIP-Seq is a method of analyzing the transcriptome by combining chromatin IP with DNA sequencing. 1. Shear chromatin, and expose to a protein specific antibody. 2. Perform Immune Precipitation - Pull down all antibody bound chromatin. 3. Reverse antibody binding and excise DNA from chromatin. 4. Amplify DNA 5. Sequence DNA in bulk. |
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What is ChIP-chip? Explain it's mechanism. |
1. Sheer chromatin, and expose to a protein specific antibody. 2. IP pulls down all protein enriched DNA. 3. Reverse antibody binding, and excise DNA from the chromatin. 4. Amplify the DNA. 5. Fluorescently label the DNA. 6. Hybridize onto a chip/array. |
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Explain ChIP-chip results. |
If there is a high fold enrichment at the antibody-association sites in DNA, there is high protein binding at that region. If fold association is similar across antibody bound, and control areas then there is likely low protein association in that DNA region. |
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Explain ChIP-seq results. |
In ChIP-Seq the height of peaks corresponds to the number of bound reads. RNAPIII and SRC-3 have increased DNA binding (association) in the presence of estrogen. |
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Explain the mouse embryo and histone acetylaase (HAT). |
A HAT protein, p300 is observed to interact with known enhancer sites. It is found to bind at different enhancer sites at different times, and will drive local gene expression when bound. |
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How can we measure DNA copy number? |
Use Comparative Genome Hybridization (CGH). Hybridize genomic DNA instead of cDNA created from RT-PCR. Compare your array to a reference to ascertain deletions or substitutions. |
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How does CRISPR-Cas9 work? |
Cas-9 is a restriction enzyme (cleaves DNA) that can be targeted to specific sequences by a sgRNA that is complementary to your target sequence. If a novel piece of DNA is introduced along with Cas9-sgRNA, endogenous cell repair will incorporate it into the genome. |
