1. Announcements Lab notebooks due Monday by 5 No Ch. 9 Part 2 homework
We will have lab next week. It just isn’t a typical lab-discussing individual projects

2. Ch. 9 Part 2: Biotechnology and Recombinant DNA

3. Toolkit: DNA Sequencing-Why It Is Important
Human Genome Project produced great advances
Rapid growth in field of genomics
Sequencing of numerous organisms
Spawned new field of bioinformatics to analyze data
Allows determination of amino acid sequences
Comparisons of different proteins, various organisms
Evolutionary relatedness of organisms
Technology so efficient that new Human Microbiome Project aims to determine biological diversity of normal microbiota

4. Toolkit: DNA Sequencing How It Works
Dideoxy chain termination most common method
Automated and fast
In vitro DNA synthesis
Template DNA
DNA polymerase
Primer
Deoxynucleotides
dNTPs
Dideoxynucleotides
ddNTPs
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Primer 5′ 3′ T G G C C G T T G C C G T A C C G G C C C T T A A A T T G A A T 3′ 5′
Primer is added to single-stranded DNA.
DNA polymerase
Nucleotide 5′ 3′ G A C T G G C C G T T T G C C G T A C C G G C C C T T A A A T T G A A T 3′ 5′
Primer anneals to complementary sequence, serving as the nucleic acid
fragment to which DNA polymerase can add nucleotides. 5′ 3′ T G G C C G G G A A T T T A A C T T A T T G C C G T A C C G G C C C T T A A A T T G A A T 3′ 5′
Final product

5. Toolkit: DNA Sequencing How It Works
Dideoxy chain termination most common method (continued…)
Dideoxynucleotides (ddNTPs)
Serve as chain terminators
Lack 3′OH
Synthesis stops
Yields mixture of DNA
of different lengths
Denature DNA
Separate ssDNA via
electrophoresis
Read fluorescent label
on ddNTPs to obtain
sequence
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PO4 OH
Incorporation of a
deoxynucleotide (dNTP)
elongates the chain. A
subsequent nucleotide can
be added to the 3′OH. 5′ 3′ OH 3′ 5′
PO4 H
Incorporation of a
dideoxynucleotide (ddNTP);
the ddNTP lacks a 3′OH. 5′ 3′ OH 3′ 5′ H
PO4
Chain elongation is
terminated. No additional
nucleotides can be added
due to the lack of a 3′OH. OH 5′ X 3′ 5′

6. Toolkit: DNA Sequencing
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Key ingredients in the reaction:
• Primer and template (shown annealed)
A T A G C T A C C T A G
• DNA polymerase
• Deoxynucleotides (dATP, dTTP, dGTP, dCTP)
• Fluorescently labeled dideoxynucleotides (ddATP, ddTTP, ddGTP, ddCTP; a very small amount of each)
Gel electrophoresis
Laser reads color
Determines sequence 2
Chain elongation is terminated randomly
when DNA polymerase incorporates a
dideoxynucleotide (indicated by a colored
box and an asterisk).
Products of the reaction: 3
The fragments are denatured, and the single
strands then separated by gel electrophoresis.
The color of the fluorescent marker indicates
the terminating dideoxynucleotide.
Results:
T A T C G A T G G A T C*
A T A G C T A C C T A G
T A T C G A T G G A T*
A T A G C T A C C T A G
T A T C G A T G G A*
A T A G C T A C C T A G
T A T C G A T G G*
A T A G C T A C C T A G
T A T C G A T G*
A T A G C T A C C T A G
T A T C G A T*
A T A G C T A C C T A G
Decreasing fragment size
T A T C G A*
A T A G C T A C C T A G
T A T C G*
A T A G C T A C C T A G
T A T C*
A T A G C T A C C T A G
T A T*
A T A G C T A C C T A G
T A*
A T A G C T A C C T A G T*
A T A G C T A C C T A G

7. Toolkit: Polymerase Chain Reaction (PCR)
Allows amplification to millions of copies of DNA
Matter of hours
Products can be visualized
via gel electrophoresis
Allows detection of
specific sequences
Can detect organisms
without culturing
E.g., pathogens
Requires template DNA,
polymerase, primers,
deoxynucleotides
(dATP, dGTP, dCTP, dTTP)
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DNA from Patient A
DNA from Patient B
PCR amplifies
specific sequence
of interest.
No DNA amplified
Gel electrophoresis of
PCR amplified samples
Patient A
Patient B
Conclusion:
Patient A is positive (infected);
Patient B is negative.

8. Toolkit: Polymerase Chain Reaction (PCR)
Three-step amplification cycle
DNA denatured by heating (~95°C)
Temperature lowered (~50°C) to allow primers to anneal
Temperature raised (~70°C) to allow DNA synthesis
DNA doubled in each cycle, so exponential increase
Heat-stable Taq polymerase from Thermus aquaticus critical
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Region of DNA to be amplified 3′ 3′ 5′ 1
Heating to 95°C denatures DNA. 5′ 3′
Primer
Primer 3′ 5′ 2
Cooling to 50°C allows the added
primers to anneal to the single-
stranded templates. 5′ 3′ 3′ 5′ 5′ 3′ 3′ 5′ 3
DNA synthesis occurs when the
temperature is raised to 72°C. 5′ 3′ 3′ 5′ 5′ 3′ 3′ 5′ 5′ 3′ 3′ 5′ 5′ 3′ 3′ 5′
The products of one 3-step cycle of PCR

9. Toolkit: Polymerase Chain Reaction (PCR)
Primers define length of PCR product
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Target DNA
Mid-length strands are synthesized
from the full-length templates. The
sites to which the primers annealed
determine the 5′ ends of these strands. 5′
Template 3′ 3′ 5′
Mid-length
fragment
Primer
Outcome of cycle 1
(as well as subsequent cycles)
Primer
Mid-length
fragment 5′ 3′ 3′ 5′
Template 2
Short fragment 5′ 3′
When the mid-length strands made
in the reaction above are used as
templates, short strands are
generated. The 3′ ends of the
fragments are complementary to the
5′ ends of the primers. 3′ 5′
Cycle 1 mid-length
fragment (template)
Outcome of cycle 2
(as well as subsequent cycles)
Cycle 1 mid-length
fragment (template) 5′ 3′ 3′ 5′
Short fragment 3
Short fragment
When short strands made in the
reaction above are used as
templates, like-sized strands are
generated. This is the double-
stranded target molecule that will be
amplified exponentially. 5′ 3′ 3′ 5′
Cycle 2 short
fragment (template)
Outcome of cycle 3
(as well as subsequent cycles)
Cycle 2 short
fragment (template) 5′ 3′
Produced when full-length molecule is used as a template 3′ 5′
Produced when mid-length molecule is used as a template
Short fragment
Produced when target fragment is used as a template

10. Toolkit: Polymerase Chain Reaction (PCR)
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Exponential amplification of DNA
Increasing numbers of product of correctly defined length
~30 cycles typical
Time 0
End of cycle 1
End of cycle 2
End of cycle 3
End of cycle 4
End of cycle 5
(2)
(8)
(22)

11. Gel Electrophoresis of PCR Product

12. Toolkit: Probe Technologies-Why Important
DNA probes locate specific nucleotide sequence
Probe is single-stranded piece of DNA
Will hybridize to complementary sequence
Labeled with marker
Numerous different
probe technologies
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Probe
Label 3′ T G G C C G T T G C C G T A C C G G C C C T T A A A T T G A A T 3′ 5′
Probe is added to single-stranded DNA that has been attached to a
solid surface. 5′ 3′ T G G C C G T T G C C G T A C C G G C C C T T A A A T T G A A T 3′ 5′
Probe anneals to complementary sequence. Because of the label it
carries, its location can easily be determined.

13. Toolkit: Probe Technologies
Colony Blotting
Allows detection of colonies that have specific sequence of DNA
Commonly used to identify which clones in a collection contain sequence of interest
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Nylon membrane
Probe bound to DNA 1
Colonies on an agar
plate. 2
Colonies are transferred
in place (“blotted”) to a
nylon membrane. 3
The membrane is
soaked in an alkaline
solution to lyse the cells
and denature their
DNA. 4
Probe is added that
binds to DNA of
interest. 5
By locating the
positions to which
probe has bound,
colonies that have the
DNA of interest can be
located.

14. Toolkit: Probe Technologies
Fluorescence in situ Hybridization (FISH)
Probe hybridizes to nucleotide sequences in intact cells fixed to microscope slide
Cells visualized via fluorescence microscope
Probe often binds to rRNA
sequences since numerous
Rapid identification in
specimen without culturing
Related organisms or
specific species

15. Toolkit: Probe Technologies
DNA Microarrays
Studies of gene expression
Also used to detect DNA sequences
mRNA is isolated, converted to labeled cDNA, hybridized to array
Array has numerous short probes specific to each gene of interest
Allows comparisons of conditions