BIO 326R 1st Edition Lecture 21 Outline of Last Lecture I. Phylogeny/Relatednessa. Morphological characteristicsII. Woese and 16s rRNAa. Sequencing 16s rDNAIII. Metagenomicsa. Microbiota testingIV. ObesityV. AntibioticsVI. Microbiota CuresOutline of Current Lecture I. Sequencinga. Sanger and Gilbert—Sanger sequencingi. Random sequencingii. Whole genome shot gun sequencingiii. Hierarchical shot gun sequencingb. IllumineII. We have the genome. Now what?a. Sequencing indidualsb. Comparative genomicsc. Personal genome identified by surnamed. sequencing 100s or 1000s of microbial genomes is easyCurrent LectureSequencing- Sanger and Gilbert—Sanger Sequencingo 1980 Nobel Prize in chemistry for Sanger sequencing start from a primer add dNTPs, DNA, polymerase, and ddNTPs- ddNTPs terminate synthesis when incorporatedThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute. mix all for ddNTPs (ddATP, ddTTP, ddGTP, ddCTP), labeled with different colors to produce a range of fragments of various lengths- ddNTP= dideoxynucleotides will add on to previous base but the next base is unable to add on to it and synthesis is terminated- ex: ddATP will terminate synthesis at an A when incorporated, ddTTP will terminate synthesis at a T when incorporated, etc. use a gel to separate and use the colors to get the sequenceo (1982) Random sequencing approach was used Stack together to align sequence and eventually obtain a whole genome from the arrangement of random sequence fragments- Arrange sequence in order by stacking regions of overlapo Whole Genome shot gun sequencing Take apart the whole genome at once, determine sequence of the fragments, put the entire genome back togethero Hierarchical shot gun sequencing Take apart pieces of the genome, put together the fragments of the pieces, put the pieces together, until the entire genome is in order- Genome Sequencing of various specieso (1995) Haemophilus influenzae 1.8 million basepairs Bacterium that was the first free living organism to be completely sequenced with whole genome shot gun sequencingo (1996) Saccharomyces cerevisiae Yeast 12 million bps One of the 1st eukaryotes sequencedo (1998) C. elegans Nematodeo (2000) Drosophila melangastor 120 million bpso (2001) Human genome project “draft” sequence of 3 billion base pairs Performed by shotgun method So many base pairs, but not so many genes 16 x’s more bps than nematode, but about the same number of genes- Illuminao 180 million reads/lane * 200 bp/read= 36 bil bp/laneo 36 bil bp/lane * 8 lanes/run = ~300 bil bp/runo 300 bil bp/run / 3GB human genome= 100 human genome/runWe have the genome. Now what?- Sequence cost has decreased each year- Opens possibility of sequencing of individualso The future of medicine! Can sequence and determine what genes an individual carries- Facilitates comparative genomics- Personal genome identified by surnameo Can use sequence to find out who someone is- For microbiology, sequencing 100s or 1000s of microbial genomes is
View Full Document
Unlocking...