Housekeeping

1
Housekeeping

• Homework 3 due Monday
• Questions 1-4, 6, 8
• SKIP QUESTION 9
• Exam III-next Friday!
• Review session-Cant due Tuesday!
• Monday or Thursday evening?
• Covers Ch13, Ch12, Ch8, Ch9 (through Monday)
• Cancer assignment due May 14th
• Lab
• Take plates out of incubator!
• Inoculate 4ml LB-Amp broth day before lab
• Quiz next week, lab notebooks due May 12th

2
Forensics
3
Variation in gene sequence
OUR CHROMOSOMES
GENE
Humans Introns
Regulatory sequence
Noncoding sequence
Sequence variation
4
DNA polymorphisms
Alteration in DNA sequence
5
Polymorphic Genes When two or more alleles are
present 1 of population (more than one wild
type allele)
NO EFFECT ON PHENOTYPE
6
DNA polymorphisms
DNA markers
Identify differences/similarities in organisms
7
What sort of DNA polymorphisms are there??
(primarily eukaryotes)
8
DNA polymorphisms
Class
Cause
Definition
9
Microsatellites
AAAAAAAAAAAAAAAAAAA
ATATATATATATATATATAT
CAGCAGCAGCAGCAGCAG

• Many different alleles based on of repeats
• Due to replication slippage

10
Fig. 9.3
11
Fig. 9.3
12
DNA polymorphisms
Class
Cause
Definition
SNPs
mutations
Single Nucleotide Polymorphisms
Microsatellites
Replication slippage
15-100 tandem repeats of 2 or 3 bp sequence
13
Minisatellites
ATACGCTCGCGA ATACGCTCGCGA ATACGCTCGCGA
ATACGCTCGCGA
10-40 bp repeat

• Many different alleles based on of repeats
• Due to unequal crossing over

14
Fig. 9.4
15
DNA polymorphisms
Class
Cause
Definition
SNPs
mutations
Single Nucleotide Polymorphisms
Microsatellites
Replication slippage
15-100 tandem repeats of 2 or 3 bp sequence
Minisatellites VNTR
Unequal crossovers
10-1000s tandem repeats of 10-40 bp sequence
16
What can we do with DNA polymorphisms?
17
Forensics
18
Want a DNA snapshot
DNA fingerprint
19
How likely is it that 2 unrelated persons will
have the same DNA fingerprint at one loci with
2 w.t. alleles?
20
At one loci with 2 w.t. alleles probability of 2
people with same genotype
Fig. 9.20
All parents heterozygotes
At multiple loci??
21
DNA fingerprint of DNA polymorphisms
22
Detection of DNA polymorphisms

• RFLP
• RFLP PCR
• PCR Southern
• RAPD

23
DNA Fingerprint-RFLP analysis
Restriction Fragment Length Polymorphism

• 1. Digest genomic DNA
• 2. Design probe for a genetic marker
• 3. Southern hybridization

24
Digest genomic DNA
Use restriction enzymes
25
Restriction digest
Fig. 9.5
VARIATION IN RESTRICTION SITES
26
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

27
Design DNA probe
Design for region where polymorphisms are present
(different restriction sites)
28
Fig. 9.5
29
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
30
Fig. 9.5
Detect variation at each locus!
31
Do micro/minsatellites create additional or
delete restriction sites?
32
Forensics RFLP at
minisatellites
Restriction digest outside minisatellites
(minisatellites at multiple sites!!)
Fig. 9.13
See variance in fragment size (not of
restriction sites)
PROBE??
33
Forensics RFLP at
minisatellites
Restriction digest outside minisatellites
(minisatellites at multiple sites!!)
Fig. 9.13
34
Forensics RFLP at
minisatellites
Southern hybridization with minisatellite probe
Fig. 9.13
35
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