DNA The Genetic Material

Transcription

1 DNA The Genetic Material Replication, Amplification, and Sequencing Scientific History The march to understanding that DNA is the genetic material T.H. Morgan (1908) Frederick Griffith (1928) Avery, McCarty & MacLeod (1944) Hershey & Chase (1952) Watson & Crick (1953) Meselson & Stahl (1958) Genes are on chromosomes T.H. Morgan working with Drosophila (fruit flies) genes are on chromosomes but is it the protein or the DNA of the chromosomes that are the genes? through 1940 proteins were thought to be genetic material Why? The Transforming Factor Frederick Griffith Streptococcus pneumonia bacteria was working to find cure for pneumonia harmless live bacteria mixed with heat-killed infectious bacteria causes disease in mice substance passed from dead bacteria to live bacteria = Transforming Factor 1928 The Transforming Factor live pathogenic strain of bacteria mice die live non-pathogenic strain of bacteria mice live heat-killed pathogenic bacteria A. B. C. D. mice live mix heat-killed pathogenic & non-pathogenic bacteria mice die Transformation? something in heat-killed bacteria could still transmit disease-causing properties 1944 DNA is the Transforming Factor Avery, McCarty & MacLeod purified both DNA & proteins from Streptococcus pneumonia bacteria which will transform non-pathogenic bacteria? injected protein into bacteria no effect injected DNA into bacteria transformed harmless bacteria into virulent bacteria

2 Avery, McCarty & MacLeod Oswald Avery Colin MacLeod Maclyn McCarty Confirmation of DNA Hershey & Chase classic blender experiment worked with bacteriophage viruses that infect bacteria grew phage viruses in 2 media, radioactively labeled with either 35 S in their proteins 32 P in their DNA infected bacteria with labeled phages Hershey & Chase Hershey & Chase Protein coat labeled with 35 S T2 bacteriophages are labeled with radioactive isotopes S vs. P DNA labeled with 32 P Which radioactive marker is found inside the cell? Which molecule carries viral genetic info? bacteriophages infect bacterial cells bacterial cells are agitated to remove viral protein coats Martha Chase Alfred Hershey 35 S radioactivity found in the medium 32 P radioactivity found in the bacterial cells Blender Experiment Radioactive phage & bacteria in blender 35 S phage radioactive proteins stayed in supernatant therefore protein did NOT enter bacteria 32 P phage radioactive DNA stayed in pellet therefore DNA did enter bacteria Confirmed DNA is transforming factor

3 Chargaff DNA composition: Chargaff s rules varies from species to species all 4 bases not in equal quantity bases present in characteristic ratio humans: A = 30.9% T = 29.4% G = 19.9% C = 19.8% 1947 Structure of DNA Watson & Crick developed double helix model of DNA other scientists working on question: Rosalind Franklin Maurice Wilkins Linus Pauling Franklin Wilkins Pauling Watson and Crick Rosalind Franklin ( ) Double Helix Structure of DNA Directionality of DNA You need to number the carbons! it matters! PO 4 nucleotide N base 5 CH 2 O 4 ribose 1 the structure of DNA suggested a mechanism for how DNA is copied by the cell 3 OH 2

4 The DNA Backbone Putting the DNA backbone together refer to the 3 and 5 ends of the DNA the last trailing carbon PO 4 5 base CH 2 O O C O P O O base CH 2 O OH 3 Base Pairing in DNA Purines adenine (A) guanine (G) Pyrimidines thymine (T) cytosine (C) Pairing A : T C : G Anti-parallel Strands Phosphate to sugar bond involves carbons in 3 & 5 positions DNA molecule has direction complementary strand runs in opposite direction It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material. Watson & Crick Bonding in DNA phosphodiester bonds hydrogen bonds strong or weak bonds? How do the bonds fit the mechanism for copying DNA? 5 Copying DNA Replication of DNA base pairing allows each strand to serve as a pattern for a new strand Models of DNA Replication Alternative models so how is DNA copied?

5 1958 Semi-conservative Replication Meselson & Stahl label nucleotides of parent DNA strands with heavy nitrogen = 15 N label new nucleotides with lighter isotope = 14 N Semi-conservative replication Make predictions 15 N strands replicated in 14 N medium 1st round of replication? 2nd round? The Most Elegant Experiment in Biology parent replication DNA Replication Large team of enzymes coordinates replication Replication: 1st step Unwind DNA helicase enzyme unwinds part of DNA helix at ori forms replication forks stabilized by single-stranded binding proteins single-stranded binding proteins Replication: 2nd step Bring in new nucleotides to match up to template strands Energy of Replication Where does the energy for the bonding come from? energy GTP TTP CTP ATP GMP TMP CMP ADP AMP single-stranded binding proteins

6 Energy of Replication The nucleotides arrive as nucleosides DNA bases with P P P DNA bases arrive with their own energy source for bonding bonded by DNA polymerase III Replication Adding bases can only add nucleotides to 3 end of a growing DNA strand strand grow 5' 3 5' energy energy energy DNA P III 3' ATP GTP TTP CTP energy 3' leading strand 5' Priming DNA Synthesis DNA polymerase III can only extend an existing DNA molecule cannot start new one cannot place first base short RNA primer is built first by primase starter sequences DNA polymerase III can now add nucleotides to RNA primer Leading & Lagging Strands Leading strand - continuous synthesis Okazaki Lagging strand - Okazaki fragments - joined by ligase - spot welder enzyme Okazaki Fragments Cleaning Up Primers DNA polymerase I removes sections of RNA primer and replaces with DNA nucleotides

7 Replication Enzymes helicase DNA polymerase III primase DNA polymerase I ligase single-stranded binding proteins And in the end Ends of chromosomes are eroded with each replication an issue in aging? ends of chromosomes are protected by telomeres Telomeres Expendable, non-coding sequences at ends of DNA short sequence of bases repeated 1000s times TTAGGG in humans Telomerase enzyme in certain cells enzyme extends telomeres prevalent in cancers Why? Replication Bubble Adds 1000 bases/second! Which direction does DNA build? List the enzymes & their role DNA Polymerase Review DNA polymerase III 1000 bases/second main DNA building enzyme DNA polymerase I 20 bases/second editing, repair & primer removal DNA polymerase III enzyme Editing & Proofreading DNA 1000 bases/second = lots of typos! DNA polymerase I proofreads & corrects typos repairs mismatched bases excises abnormal bases repairs damage throughout life reduces error rate from 1 in 10,000 to 1 in 100 million bases

8 Fast & Accurate! It takes E. coli <1 hour to copy 5 million base pairs in its single chromosome divide to form 2 identical daughter cells Human cell copies its 6 billion bases & divide into daughter cells in only few hours remarkably accurate only ~1 error per 100 million bases ~30 errors per cell cycle What s it really look like? The Central Dogma flow of genetic information within a cell transcription DNA replication RNA translation protein Polymerase Chain Reaction (PCR) What if you have to copy DNA with not a lot to begin with? PCR is a method for making many copies of a specific segment of DNA ~only need 1 molecule of DNA to start PCR Process PCR Process It s copying DNA in a test tube! What do you need? template strand DNA polymerase enzyme nucleotides primer Thermocycler

9 Kary Mullis development of PCR technique a copying machine for DNA PCR Process What do you need to do? in tube: DNA, enzyme, primer, nucleotides heat (90 C) DNA to separate strands (denature) cool to hybridize (anneal) & build DNA (extension) The Polymerase Problem Heat DNA to denature it 90 C destroys DNA polymerase have to add new enzyme every cycle almost impractical! Need enzyme that can withstand 90 C Taq polymerase from hot springs bacteria Thermus aquaticus PCR Primers The primers are critical! need to know a bit of sequence to make proper primers primers bracket target sequence start with long piece of DNA & copy a specified shorter segment primers define section of DNA to be cloned cycles 3 steps/cycle 30 sec/step DNA Sequencing Sanger method determine the base sequence of DNA dideoxynucleotides ddatp, ddgtp, ddttp, ddctp missing O for bonding of next nucleotide terminates chain DNA Sequencing Sanger method synthesize complementary DNA strand in vitro in each tube: normal N-bases dideoxy N-bases dda, ddc, ddg, ddt DNA polymerase primer 2 buffers & salt

10 Reading the Sequence Load gel with sequences from dda, ddt, ddc, ddg in separate lanes read lanes manually & carefully polyacrylamide gel Fred Sanger This was his 2nd Nobel Prize!! 1st was in 1958 for the structure of insulin Advancements to Sequencing Fluorescent tagging no more radioactivity all 4 bases in 1 lane each base a different color Automated reading Advancements to Sequencing Fluorescent tagging sequence data Computer read & analyzed Advancements to Sequencing Capillary tube electrophoresis no more pouring gels higher capacity & faster Big labs! economy of scale Applied Biosystems, Inc (ABI) built an industry on these machines 384 lanes PUBLIC Joint Genome Institute (DOE) MIT Washington University of St. Louis Baylor College of Medicine Sanger Center (UK) PRIVATE Celera Genomics

11 Automated Sequencing Machines Really BIG labs! Human Genome Project 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 Different Approaches gov t method map-based method 1. Cut chromosomal DNA segment into fragments, arrange based on overlapping nucleotide sequences, and clone fragments. 2. Cut and clone into smaller fragments. 3. Assemble DNA sequence using overlapping sequences. Craig Venter s method shotgun method 1. Cut DNA from entire chromosome into small fragments and clone. 2. Sequence each segment & arrange based on overlapping nucleotide sequences. Human Genome Project 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 Sequence of 46 Human Chromosomes Raw Genome Data 3G of data 3 billion base pairs

12 Nucleotide substitutions Dec-82 Dec-83 Dec-84 Dec-85 Dec-86 Dec-87 Dec-88 Dec-89 Dec-90 Dec-91 Dec-92 Dec-93 Dec-94 Dec-95 Dec-96 Dec-97 Dec-98 Dec-99 Dec-00 Dec-01 Dec-02 Jun-03 Colonie High BIOCHEMISTRY/MOLECULAR BIOLOGY GenBank Organizing the Data Database of genetic sequences gathered from research Publicly available! And we didn t stop there The Progress 4.E+10 3.E+10 3.E bacterial genomes 2.E+10 first metazoan complete (flatworm) 2.E+10 1.E+10 5.E+09 0.E+00 # of DNA base pairs (billions) in GenBank first eukaryote complete (yeast) First 2 bacterial genomes complete Data from NCBI and TIGR ( and ) Official 15 year Human Genome Project: eukaryotic genomes complete or near completion including Homo sapiens, mouse and fruit fly S1 How does our genome stack up? Organism Genome Size (bases) Estimated Genes Human (Homo sapiens) 3 billion 30,000 Laboratory mouse (M. musculus) 2.6 billion 30,000 Mustard weed (A. thaliana) 100 million 25,000 Roundworm (C. elegans) 97 million 19,000 Fruit fly (D. melanogaster) 137 million 13,000 Yeast (S. cerevisiae) 12.1 million 6,000 Bacterium (E. coli) 4.6 million 3,200 Human Immunodeficiency Virus (HIV) you will certainly find something! Horse/ donkey Sheep/ goat Goat/cow Rabbit/ rodent Llama/ cow Pig/ cow Dog/ cow Horse/cow Human/kangaroo Human/ cow Human/rodent Millions of years ago