Download Intro to DNA Sequencing

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
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
A.
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?
A. Generate taxonomic data to determine
evolutionary relationships
B. Mapping Genomes
C. Confirming the presence of a particular gene in
an environmental sample
D. 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.
A.
B.
C.
D.
Bioluminescent
Radioactive
Fluorescent
Intercalated
Timeline for Human Genome
Project
Year
# of fragments
# years for HGP
mapped & sequenced
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
END
Resources

http://www.dnai.org/text/mediashowcase
/index2.html?id=608

http://www.dnalc.org/ddnalc/resources/c
ycseq.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