Chapter 23 Part 2'

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• Chapter 23 Part 2.
• Biotechnology
• Transgentic animals
• Genetic engineering in plants
• PCR polymerase chain reaction
• Site-directed mutagenesis
• DNA and forensics
• Sequencing DNA

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• Transgenic
• animals

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• Genetic
• engineering in plants.

Agarobacterium tumifaciens
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• PCR
• Polymerase chain
• reaction

C F
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Cyce I
1. Heat and separate strands. 2. Add primer
100x excess cool 3. Add taq polymerase 4
dNTPs ? synthesize complementary strands
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Cycle 2
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Cycle 3
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4. Site-directed mutagenesis Use PCR to make
large amounts of a protein with a particular
mutation. You have isolated and clones a protein
that you hypothesize is a tyrosine protease
has a tyrosine (tyr) at the active site. You
examine the DNA sequence until you fine a
sequence that looks like the active site of a
tyrosine protease.
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Ser Asp Gly Tyr Val His Ile
5--//--TCAGACGGTTACGTACATATC--//-3 3--//--AGTCT
GCCAATGCATGTATAG--//-5
5--//--TCAGACGGTTTCGTACATATC--//-3
3--//--AGTCTGCCAATGCATGTATAG--//-5
In the primer A is changed to T.
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In the next round of PCR
5--//--TCAGACGGTTTCGTACATATC--//-3
3--//--AGTCTGCCAAAGCATGTATAG--//-5
Tyr? Phe (phenylalanine)
Express in a vector and check activity.
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5. DNA and forensics A. Uses B. DNA
fingerprinting C. Restriction Fragment
Lerngth Polymorphism (RFLP)
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A. Uses

• In cases of crimes such as rape to
• establish guilt or innocence.
• Paternity
• DNA samples are taken from members
• of the armed forces.

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B. DNA fingerprinting.
1. Make restriction fragments.
2. Run on a gel
C F
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C. Restriction Fragment Lerngth Polymorphism
(RFLP)
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6. Determining the base sequence of DNA.
Step 1. Put a small amount of dideoxy NTPs in
the replication mixture.
CF
Terminates the chain at random positions.
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Template 3CATGCTCGACGA5 Primer 5-GT
dATPddATP dATP dATP dATP dCTP
dCTP dCTPddCTP dCTP dGTP
dGTPddGTP dGTP dGTP dTTP dTTP
dTTP dTTPddTTP
Four different reaction mixtures. Each has one of
the dideoxy NTPs
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Template 3CATGCTCGACGA5 Primer 5-GT
dATPddATP dATP dATP dATP dCTP
dCTP dCTPddCTP dCTP dGTP
dGTPddGTP dGTP dGTP dTTP dTTP
dTTP dTTPddTTP

?
GTA GTATGCA
GTACCAG GTACCAGCTG
GTAC GTACC GTACCAGC GTACCAGCTGC

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GTA GTATGCA
GTACCAG GTACCAGCTG
GTAC GTACC GTACCAGC GTACCAGCTGC