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Dna sequencing
- 1. DNA Sequencing Tai Kao-Sowa Stanford University
- 2. What is it? DNA -> RNA -> amino acid sequences -> proteins Methylation/Acetylation RNA Processing RNA Silencing Proteins do stuff
- 3. Why do we care? Diagnose disease Synthesize DNA copies for analysis Learn how to produce new substrates Mass produce important proteins Genetically modify organisms Genetically modify humans Understand how the body works Forensics Understanding human history Condensed information storage Synthetic computation Synthetic organisms Cure genetic diseases Individualized medicine
- 5. Wobble & Redundancy tRNA can bond to multiple amino acids Multiple start codons, “special cases” GGU, GGC, GGA, GGG all code for glycine
- 6. More variability Codon (AUG start, UAG, UGA, UAA stop) Reading Frames 20 amino acids: RNA Splicing Epigenetics
- 7. History Experimental sequencing -Random radioactive nucleotides fill end sequences -Bacteriophages Sanger sequencing Automation, whole genome shotgun sequencing Next generation “massive parallelization”
- 8. Variable Length Ends ddNTP lack 3’ hydroxyl
- 9. Shotgun Sequencing Cleaved into manageable sequences Move to capillary electrophoresis 96 parallel
- 10. Next-Gen Sequencing Wells, beads, and nanopores Tradeoff of size/accuracy for massive parallelization Still (mostly) use terminators to find a base at a time
- 11. Illumina Sequencing Short 100-150 bp reads Oligonucleotide chip Cluster generation Fluorescently labeled nucleotides Terminator limits to 1 bond Take picture, remove terminator Rinse and repeat
- 12. Roche 454 (Pyrosequencing) DNA fragmented PCR amplification Single type of nucleotide, releases light Intensity of reaction signals # Rinse and repeat 20,000,000 bp/run
- 13. Ion Torrent Sequencing DNA fragmented PCR amplification Bead into well Binding of nucleotide changes pH Add one type of nucleotide Lower pH = more bonds
- 14. Next-Gen Sequencing Large computational power Amplification and parallel reading of many strands (Illumina can process 1 human genome/hour/instrument) DNA nanoballs, nanopore, electron tunneling, etc. Longreads We’ve hit the $1k human genome ($100 next) https://www.ebi.ac.uk/training/online/course/ebi-next-generation-sequencing-practical-course
- 15. Pros Cons DNA as Storage -Easy replication -Parallel access -Possibility for biological real- time error checking -1 gram of DNA is 108 terrabytes (1021 bases) -No power requirements -Comparatively difficult to modify -Difficult to access -Everything is slow -Duplication -Access -Modifications Easily destroyed/mutated
- 16. Takeaways DNA sequencing is transitioning to a medium stage technology DNA has potential -Extremely small size -Direct interface with biological products -Storage time is on order of 100s of years -Molecules work independently -Parallel processing -Messy