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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

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Intro to DNA Sequencing


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definition
Definition
  • A technique used to determine the sequence of nucleotide bases in a DNA molecule or fragment
quick quiz
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
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
Non-examples
  • DNA sequencing is NOT commonly used for…
    • DNA fingerprinting
    • Making monoclonal antibodies
    • Conducting stem cell research
quick quiz6
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
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
technique 2 sanger dideoxy sequencing
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
technique 3 dye terminator 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
slide12

Dye-Termination Sequencing

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

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

quick quiz14
Quick Quiz

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

  • Bioluminescent
  • Radioactive
  • Fluorescent
  • Intercalated
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

Whole Genome Sequencing
resources
Resources
  • http://www.dnai.org/text/mediashowcase/index2.html?id=608
  • http://www.dnalc.org/ddnalc/resources/cycseq.html
california state chemistry standards
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
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
California State Investigation and ExperimentationStandards

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
National Standards

Grades 6-12

Content Standard A: Science as Inquiry

Content Standard C: Life Science

Content Standard E: Science and Technology