MBB 1 DNA Fingerprinting Handout

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

MBB 1 DNA Fingerprinting Handout

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