1. Intro to DNA Sequencing

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

3. 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

4. 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

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

6. 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

7. 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

9. 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

10. Sanger Sequencing

11. 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

12. Dye-Termination Sequencing

13. Dye-terminator sequencing

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

15. 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)

16. 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

17. END

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

19. 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

20. 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

21. 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

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