Intro to DNA Sequencing

Definition A technique used to determine the sequence of nucleotide bases in a DNA molecule or fragment

Quick Quiz DNA sequencing is used to: Determine the sequence of nucleotide bases in a molecular project Determine the order of genes in a chromosome Determine whether or not PCR was successful Determine whether or not DNA purification was successful

Application Examples DNA sequencing is commonly used to… Identify useful regions of DNA for other applications Identify nucleotides in a gene and the inferred protein sequence Generate data for taxonomic analysis Identify gene defects associated with certain diseases Analyze whole genomes

Non-examples DNA sequencing is NOT commonly used for… DNA fingerprinting Making monoclonal antibodies Conducting stem cell research

Quick Quiz Which of the following is NOT an application of DNA sequencing? Generate taxonomic data to determine evolutionary relationships Mapping Genomes Confirming the presence of a particular gene in an environmental sample DNA fingerprinting

Technique 1: Maxam-Gilbert (chemical cleavage) A pool of identical DNA fragments is labeled at one end and split into four tubes The fragments are broken preferentially at one of the four nucleotides, under conditions where only an average of about one break is made per fragment The 4 reactions are denatured and run out on an acrylamide sequencing gel

http://www.cbs.dtu.dk/staff/dave/roanoke/genetics980211.html

Technique 2: Sanger (dideoxy sequencing) The reaction is split into 4 tubes, each containing… Template DNA, primer, DNA polymerase, dNTPs (A, T, G, C), and a low concentration of one dideoxynucleotide (ddA, ddT, ddG, or ddC) Template DNA is denatured, a primer anneals, and a complementary DNA strand is extended by DNA polymerase Once incorporated, a ddNTP will block further DNA extension This results in each reaction tube containing a mixture of DNA strands of many different lengths The 4 reactions are denatured and run in separate lanes of an acrylamide sequencing gel

Sanger Sequencing

Technique 3: Dye-terminator sequencing Combines the Sanger method (chain termination) with PCR ddNTPs are fluorescently labeled so each one has a unique chromaphore Gel is read by a sequencer, which translates the DNA bands into a 4-color chromatogram

Dye-Termination Sequencing http://oregonstate.edu/instruction/bb492/figletters/FigG4.html http://www.igh.cnrs.fr/equip/sequence/DNASEQCENTER.html

Dye-terminator sequencing

Quick Quiz In Dye-Terminator sequencing, each ddNTP is labeled with a unique _________ tag. Bioluminescent Radioactive Fluorescent Intercalated

Timeline for Human Genome Project Year # of fragments mapped & sequenced # years for HGP at that rate 1967 none not possible yet 1977 3 4,000,000 1987 12 1000 1997 30,000 50 2007 All 13 (actual)

Whole Genome Sequencing Examples of those sequenced by 2007: Human Sea urchin Chimpanzee Mouse Rat Honey bee 2 puffer fish 2 fruit flies 2 sea squirts 2 roundworms Baker's yeast Escherichia coli More recently: Horse Pig Chicken Dog Rhesus macaque monkey Over 300 sequenced to date - many are bacteria

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Resources http://www.dnai.org/text/mediashowcase/index2.html?id=608 http://www.dnalc.org/ddnalc/resources/cycseq.html

California State Chemistry Standards Grade 8 5a. Reactant atoms and molecules interact to form products with different chemical properties 6c. Living organisms have many different kinds of molecules…including DNA Grades 9-12 2. Biological, chemical and physical properties of matter result from the ability of atoms to form bonds from electrostatic forces… 6. Solutions are homogenous mixtures of two or more substances 8. Chemical reaction rates depend on factors that influence the frequency of collision of reactant molecules

California State Biology Standards Grade 7 2e. DNA is the genetic material of living organisms and is located in the chromosomes of each cell 3a. Genetic variation and environmental factors are causes of evolution and diversity of organisms Grades 9-12 1. Fundamental life processes of plants and animals depend on a variety of chemical reactions that occur in specialized areas of the cell 2. Mutation and sexual reproduction lead to genetic variation in a population 4. Genes are a set of instructions encoded in the DNA sequence of each organism… 5. Genetic composition of cells can be altered by incorporation of exogenous DNA into the cells

California State Investigation and Experimentation Standards Grades 7 b. Select and use appropriate tools and technology Grade 8 a. Plan and conduct a scientific investigation to test a hypothesis Grades 9-12 a. Select and use appropriate tools and technology to perform tests, collect data, analyze relationships, and display data b. Identify and communicate sources of unavoidable experimental error c. Identify possible reasons for inconsistent results d. Formulate explanations by using logic and evidence k. Recognize the cumulative nature of scientific evidence l. Analyze situations and solve problems that require combining and applying concepts from more than one area of science

National Standards Grades 6-12 Content Standard A: Science as Inquiry   Content Standard C: Life Science Content Standard E: Science and Technology