A Lot More Advanced Biotechnology Tools Sequencing
* Sanger method (Fig ) ● determine the base sequence of DNA ● based on replication ● dideoxyribonucleotides ddATP, ddGTP, ddTTP, ddCTP missing O for bonding of next nucleotide terminates the growing chain DNA S EQUENCING
* Sanger method ● synthesize complementary DNA strand in vitro ● in each tube: “normal” N-bases Tagged dideoxy N-bases ● ddA, ddC, ddG, ddT DNA polymerase primer buffers & salt
* R EADING THE SEQUENCE Load gel with sequences from ddA, ddT, ddC, ddG in separate lanes ● read lanes manually & carefully ● polyacrylamide gel
* F RED S ANGER 1978 | 1980 This was his 2nd Nobel Prize!! ● 1st was in 1958 for the structure of insulin
* A DVANCEMENTS TO SEQUENCING Fluorescent tagging ● no more radioactivity ● all 4 bases in 1 lane each base a different color Automated reading
* A DVANCEMENTS TO SEQUENCING Fluorescent tagging sequence data Computer read & analyzed
* Applied Biosystems, Inc (ABI) built an industry on these machines A DVANCEMENTS TO SEQUENCING Capillary tube electrophoresis ● no more pouring gels ● higher capacity & faster 384 lanes
* PUBLIC Joint Genome Institute (DOE) MIT Washington University of St. Louis Baylor College of Medicine Sanger Center (UK) PRIVATE Celera Genomics Big labs! ● economy of scale
* A UTOMATED S EQUENCING MACHINES Really BIG labs!
* H UMAN G ENOME P ROJECT U.S government project ● begun in 1990 estimated to be a 15 year project ● DOE & NIH initiated by Jim Watson led by Francis Collins ● goal was to sequence entire human genome 3 billion base pairs Celera Genomics ● Craig Venter challenged gov’t ● would do it faster, cheaper ● private company
* D IFFERENT APPROACHES 3. Assemble DNA sequence using overlapping sequences. “map-based method” gov’t method “shotgun method” Craig Venter’s method 1. Cut DNA entire chromosome into small fragments and clone. 2. Sequence each segment & arrange based on overlapping nucleotide sequences. ● Cut DNA segment into fragments, arrange based on overlapping nucleotide sequences, and clone fragments. 2. Cut and clone into smaller fragments.
* H UMAN G ENOME P ROJECT On June 26, 2001, HGP published the “working draft” of the DNA sequence of the human genome. Historic Event! ● blueprint of a human ● the potential to change science & medicine
* S EQUENCE OF 46 H UMAN C HROMOSOMES 3 billion base pairs 3G of data
* TACGCACATTTACGTACGCGGATGCCGCGACT ATGATCACATAGACATGCTGTCAGCTCTAGTA GACTAGCTGACTCGACTAGCATGATCGATCAG CTACATGCTAGCACACYCGTACATCGATCCTG ACATCGACCTGCTCGTACATGCTACTAGCTAC TGACTCATGATCCAGATCACTGAAACCCTAGA TCGGGTACCTATTACAGTACGATCATCCGATC AGATCATGCTAGTACATCGATCGATACTGCTA CTGATCTAGCTCAATCAAACTCTTTTTGCATCA TGATACTAGACTAGCTGACTGATCATGACTCT GATCCCGTAGATCGGGTACCTATTACAGTACG ATCATCCGATCAGATCATGCTAGTACATCGAT CGATACTGCTACTGATCTAGCTCAATCAAACT CTTTTTGCATCATGATACTAGACTAGCTGACT GATCATGACTCTGATCCCGTAGATCGGGTACC TATTACAGTACGATCATCCGATCAGATCATGC TAGTACATCGATCGATACT human genome 3.2 billion bases
* R AW GENOME DATA
* NCBI G EN B ANK Database of genetic sequences gathered from research Publicly available on Web!
* O RGANIZING THE DATA
* M APS OF HUMAN GENES … Where the genes are… ● mapping genes & their mutant alleles
* D EFINING A GENE … “Defining a gene is problematic because… one gene can code for several protein products, some genes code only for RNA, two genes can overlap, and there are many other complications.” – Elizabeth Pennisi, Science 2003 gene polypeptide 1 polypeptide 2 polypeptide 3 protein gene It’s hard to hunt for wabbits, if you don’t know what a wabbit looks like. RNA gene
* A ND WE DIDN ’ T STOP THERE …
* T HE P ROGRESS First 2 bacterial genomes complete 122+ bacterial genomes Data from NCBI and TIGR ( gov and ) first eukaryote complete (yeast) first metazoan complete (flatworm) 17 eukaryotic genomes complete or near completion including Homo sapiens, mouse and fruit fly Official “15 year” Human Genome Project: # of DNA base pairs (billions) in GenBank
* H OW DOES THE HUMAN GENOME STACK UP ? Organism Genome Size (bases) Estimated Genes Human (Homo sapiens) 3 billion30,000 Laboratory mouse (M. musculus) 2.6 billion30,000 Mustard weed (A. thaliana) 100 million25,000 Roundworm (C. elegans) 97 million19,000 Fruit fly (D. melanogaster) 137 million13,000 Yeast (S. cerevisiae) 12.1 million6,000 Bacterium (E. coli) 4.6 million3,200 Human Immunodeficiency Virus (HIV) 97009
* W HAT HAVE WE FOUND ? When you go looking…
* … YOU WILL CERTAINLY FIND SOMETHING !