Housekeeping

1
Housekeeping

• Homework 3 due today
• Exam III Friday!
• Review session Wednesday 5 pm (Sc278)
• Covers Ch13, Ch12, Ch8, Ch9 (through today)
• Cancer assignment due May 14th
• Lab
• Inoculate 4ml LB-Amp broth day before lab (in
  refrigerator)
• Quiz this week
• lab notebooks due Monday, May 12th

2
DNA Fingerprint-RFLP analysis
Restriction Fragment Length Polymorphism
1. Digest genomic DNA VARIATION IN RESTRICTION
SITES 2. Design probe for a genetic marker 3.
Southern hybridization
3
Disadvantages of RFLP

• Need A LOT of DNA!
• Need A LOT of time!
• Need to have a specific probe
• KNOW SEQUENCE
• Alterations to RFLP make DNA fingerprinting easier

4
Detection of DNA polymorphisms

• RFLP
• RFLP PCR
• PCR Southern
• RAPD

5
RFLP PCR

• 1. Amplify region of polymorphism
• 2. Restriction digest
• 3. Gel electrophoresis

6
RFLP PCR
Fig. 9.7
Not a lot of DNA needed
Not a lot of time
No Southern necessary
Still need to KNOW a polymorphic region
7
What if the polymorphism doesnt create a
different restriction site?
8
The genetic counselor
Will your baby have sickle cell anemia??
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Sickle cell anemia

• Abnormal red blood cells
• Genetic disease
• Mutant recessive allele
• Identified a point mutation

GENOTYPING
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Detection of DNA polymorphisms

• RFLP
• RFLP PCR
• PCR Southern
• RAPD

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PCR Southern
1. Amplify region of polymorphism 2. Southern
hybridization
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Amplify region of polymorphism

• Design primers outside of polymorphic region
• Conserved sequence

AAGCT
Allele 1
AATCT
Allele 2
13
Southern hybridization

• Probe specific for polymorphic region
• Short probe ( 20 nt)
• Allele specific oligonucleotides (ASO Probes)
• Hybridize sample twice with different probes

AAGCT
AAGCT
Allele 1
AATCT
AATCT
Allele 2
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Southern hybridization
DNA with polymorphism (has allele 1)
Allele 1 probe
Allele 2 probe
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PCR Southern

• 1. Extract DNA from sample
• 2. Divide DNA into two samples
• 3. Amplify sickle cell allele
• Primers designed for a conserved region
• 4. Southern hybridization
• ASO probes (for mutant and w.t. allele)

16
Assuming these are the two alleles, where would
you design your ASO probe? How long would it be?
Wild type allele
TGTCGAGCCAGTCCGGGTACAATCTCAGTGCGTAACTCGTACTCGATCTA
C
Mutant allele
TGTCGAGCCAGTCCGGGTACAATCACAGTGCGTAACTCGTACTCGATCTA
C
17
If the baby is a carrier for sickle cell anemia,
what would you expect the results to be? If the
baby was going to have the disease?
Wild type probe
Mutant probe
PCR of sickle cell gene in baby used in Southern
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Baby is heterozygous
Wild type probe
Mutant probe
PCR of sickle cell gene in baby used in Southern
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Disadvantages to PCR Southern

• Need TIME
• Need to identify sequence
• Polymorphic region
• Conserved exterior region

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Detection of DNA polymorphisms

• RFLP
• RFLP PCR
• PCR Southern
• RAPD

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Rapid Amplification of Polymorphic DNA RAPD

• 1. Random amplification
• 2. Gel electrophoresis

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Random amplification

• Use very short (10 nt) primers
• Amplify randomly at varied recognition sites

Fig. 9.11a
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Gel electrophoresis
Fig. 9.11
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Advantages of RAPD

• SIMPLE and FAST
• No sequence identification necessary!!
• Good for initial genome scan
• unknown organism

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Detection of DNA polymorphisms

• RFLP
• RFLP PCR
• PCR Southern
• RAPD

26
Detection of polymorphisms REVIEW

• Several different kinds of polymorphisms
• bp substitutions
• mini/microsatellites
• deletions, additions, etc..
• Several different methods to test for
  polymorphisms
• RFLP
• RFLP PCR
• PCR Southern
• RAPD
• Several different uses for polymorphisms
• Forensics
• Genotyping (genetic counseling, genetic
  screening, etc..)
• Evolution
• DNA markers for genome mapping