mbb 1 dna fingerprinting handout
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8/7/2019 MBB 1 DNA Fingerprinting Handout
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http://www.biotech.iastate.edu/biotech_
info_series/bio6.html
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The human genome contains 3 x 109 basepairs
99 - 99.9% of DNA sequence is identical fromone individual to the next
The other 0.1-1% contains highly variableregions Focus of DNA fingerprinting analysis
Used as genetic markers for DNA fingerprinting
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VNTRs Variable number tandem repeat
contain repeated sequences of 15-70 bases
STRs
Short tandem repeats contain repeated sequences of 2-4 bases
Number of repeats varies greatly between individuals.
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Restriction Fragment Length Polymorphism
(RFLP)/Southern blot
Uses VNTRs as genetic markers
Polymerase Chain Reaction (PCR)
Uses STRs as genetic markers
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1. Collect samples
2. Isolate DNA
3. Digest DNA with restriction enzyme(s) that
cut at regions flanking VNTRs
4. Separate DNA fragments
5. Transfer DNA into nylon membrane6. Target VNTR markers using DNA probes
7. Visualize and analyze DNA fingerprint
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skin
blood
semen
hair roots
saliva bones
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a difference in DNA fragment sizes after
restriction enzyme digestion
Difference results from presence of different
DNA sequences
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GAATTC
CTTAAG
EcoR1 restriction site
GACTTC
CTGAAG
Not an EcoR1 restriction site
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Technique used to separate DNA fragments by size
http://healthinformatics.wikis
paces.com/Electrophoresis
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http://www.ncbi.nlm.nih.gov/books/NBK21738/?rendertype=figure&id=A1694
Probe: Short, single-stranded, labeled (radioactive or colored) DNA orRNA that is complementary to the region of interest in the target DNA;For fingerprinting: DNA probes that specifically identify the VNTRcontaining fragments
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A technique in molecular biology used to
amplify or make more copies of a specific
piece of DNA
In vitro replication of DNA
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DNA extract (template) Contains the DNA sequence to be amplified
Nucleotides (A, T, C, G) Building blocks of DNA
Heat stable DNA polymerase Enzyme that catalyzes the polymerization of DNA Usually Taq polymerase
PCR Buffer Provides an environment suitable for the DNA polymerase activity
Primers (forward and reverse) Short, single-stranded DNAs that are complementary to the region of
interest in the template DNA
serve as starting point for DNA synthesis
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Denaturation (95°C) Separation of the template DNA strands
Annealing (55-65°C) Binding of the primers to the target DNA sequence
(via complementary base pairing)
Elongation / Extension (72°C) Synthesis of new DNA strands complementary to the
DNA template
catalyzed by DNA polymerase
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Denaturation
Annealing
Elongationhttp://oceanexplorer.noaa.gov/explorations/04etta/background/dna/media/dna_1.html
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Fast and sensitive Can be used for small amounts of DNA
Can easily amplify STRs STRs are shorter than VNTRs
Regions flanking STRs have a highly conservedsequence
Can be used for degraded DNA Even small pieces of DNA may contain STRs
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Primers Designed to amplify STRs; one primer pair for each
STR marker
When the PCR products are run on a gel, the numberof repeats for a particular STR marker can be inferredfrom the size of the PCR fragment
http://science.kennesaw.edu/~rrascati/forensicpolymorphs.htm
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After amplification, the samples are run on a
genetic analyzer machine to determine the
lengths of each amplified STR marker
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Person A: 130bp/150 bp9/14 repeats
Person B: 140bp/150bp11/14 repeats
Repeat number Length (bp)
9 131
10 135
11 139
12 143
13 147
14 151
15 155
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STR Locus Person A Person B
D19 13/10 13/13
D3 16/15 16/15
D8 14/9 14/11
TH01 6/6 Not amplified
VWA 17/16 16/16
D21 29/27 31/29
FGA 22/20 20/20
D16 11/9 6/4
D18 16/11 17/11
D2 18/19 19/18
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Forensic cases – matching suspect with evidence
Paternity testing – identifying the father
Historical investigations – forensic anthropology
Identifying missing persons
Mass disaster cases – putting pieces back
together Military DNA “dog tag” in case of casualty
Convinced felon DNA database for repeatoffenders