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

Intro to DNA SequencingIntro to DNA Sequencing

DefinitionDefinition

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

Quick QuizQuick Quiz

DNA sequencing is used to:

A. Determine the sequence of nucleotide bases in a molecular project

B. Determine the order of genes in a chromosome

C. Determine whether or not PCR was successful

D. Determine whether or not DNA purification was successful

Application ExamplesApplication 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 analysisIdentify gene defects associated with certain

diseasesAnalyze whole genomes

Non-examplesNon-examples

DNA sequencing is NOT commonly used for…

DNA fingerprintingMaking monoclonal antibodiesConducting stem cell research

Quick QuizQuick Quiz

Which of the following is NOT an application of DNA sequencing?

A. Generate taxonomic data to determine evolutionary relationships

B. Mapping GenomesC. Confirming the presence of a particular gene in

an environmental sampleD. DNA fingerprinting

Technique 1: Technique 1: Maxam-Gilbert (chemical cleavage)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: Technique 2: Sanger (dideoxy sequencing)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: Technique 3: Dye-terminator sequencingDye-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 SequencingDye-Termination Sequencing

http://oregonstate.edu/instruction/bb492/figletters/FigG4.html

http://www.igh.cnrs.fr/equip/sequence/DNASEQCENTER.html

Dye-terminator sequencing

Quick QuizQuick Quiz

In Dye-Terminator sequencing, each ddNTP is labeled with a unique _________ tag.

A. BioluminescentB. RadioactiveC. FluorescentD. Intercalated

Timeline for Human Genome Timeline for Human Genome ProjectProject

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 SequencingWhole 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

END

ResourcesResources

http://www.dnai.org/text/mediashowcase/index2.html?id=608

http://www.dnalc.org/ddnalc/resources/cycseq.html

California State Chemistry StandardsCalifornia State Chemistry Standards

Grade 85a. 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 StandardsCalifornia 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 California State Investigation and ExperimentationInvestigation and Experimentation

StandardsStandardsGrades 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 StandardsNational Standards

Grades 6-12

Content Standard A: Science as Inquiry

Content Standard C: Life Science

Content Standard E: Science and Technology