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11 Cards in this Set
- Front
- Back
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What can genome sequencing identify? |
1. Protein and non protein coding genes 2. Gene regulatory elements 3. Genome organisation and function 4. Mechanisms of genome evolution |
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What are the uses of genome sequencing? |
1. Understanding the variation between individuals between individuals 2. Characterise differences between strains / varieties / populations 3. Diagnostic assays for pathogens or disase 4. Marker assisted selection |
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Describe the hierarchical shotgun approach to genome sequencing. |
1. Physical - top down map constructed 2. A minimal number of fragments that cover the entire genome are selected for sequencing 3. Amplified genome is sheared into large pieces and cloned into a bacterial host using Bacterial artificial chromosomes 4. Due to random shearing process the clones have different ends 5. With enough coverage however a 'scaffold' of overlapping BAC contigs that cover the entire genome can theoretically be constructed |
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Describe the whole genome shotgun approach to genome sequencing. |
1. DNA sheared into random fragments 2. Fragments cloned into an appropriate vector 3. Clones sequenced from both ends - yielding two shot sequences - the end reads 4. Overlapping sequences arranged together - mate pairs / contigs 5. Sequence assembly software constructs a genome from all of the clone sequences - beginning with mate pairs to from scaffolds |
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What is the process of Sanger sequencing? |
1. The DNA to be sequenced acts a template 2. Oligonucleotide primer allows sequencing to start at a define point 3. Add small amounts of ddNTPS 4. Four separate reactions required for each template - one for each base 5. Identification of specific bases after electrophoresis |
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What is the disadvantage of sanger sequencing? |
<500 bases per day - slow and expensive |
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What are the principles of Automated - next generation - DNA sequencing technology? |
1. Sequencing reactions generate fluorescently labelled products that differ by a single nucleotide 2. Fragments are resolved on a gel 3. THe products are detected by a laser fluorescence system 4. Sequence chromatograms produced for individual clones |
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What is the generalised process for next generation sequencing? |
1. Genomic DNA is fragmented and size classes selected 2. Adapter sequences are ligated to each end of the fragment 3. Each fragment is immobilised on a solid substrate and PCR amplified to generate c.1000 copies 4. Each template sequenced sequentially 5. Incorporated bases are detected after each step and the sequence is determined |
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What are the advantages of next gen sequencing? |
1. No amplification of DNA required 2. Rapid sequencing 3. Long reads 4. Potential for sequencing modified nucleotides |
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What are the challenges which next gen sequencing must undergo? |
1. Relatively low accuracy 2. Sample prep costly 3. Sequencing of long reads is less efficient than small |
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What could you use next gen sequencing for? |
1. Sequencing complex genomes 2. Direct transcriptomes profiling |
