how many cats? a dna profiling simulation. structure of dna double helix (analogy: spiral staircase)...
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HOW MANY CATs?A DNA Profiling Simulation
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Structure of DNA• Double helix (Analogy: Spiral staircase)• Anti-parallel strands• 4 Bases (A, C, T, and G)• Complementary bases (Analogy:Puzzle pieces)• Negatively charged molecule• Organized into chromosomes in eukaryotes
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5’ 3’
5’3’
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Variable Number Tandem Repeats (VNTRs)
Discovered in 1984 by Sir Alec Jeffries
• “Junk” or Intragenic DNA that likely does not code for any protein - ~2% genome is transcribed• May be pseudogenes, retrotransposons• May be regulatory, micro RNA’s
• Short sequences (3-30 bp) repeated multiple times (10-100 times) Example: CATCATCATCAT…
• What is variable is the NUMBER of copies of the sequence in an allele3. Example: One allele might have 3 copies
[CATCATCAT] and the other allele might have 5 copies [CATCATCATCATCAT]
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•23 chromosomes from each parent = 46 total chromosomes in each child
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[CATCATCATCATCAT]Father 5[CATCATCATCATCATCATCAT]Father 7
Child’s possible VNTR alleles at this locus on chromosome 17:
1) 3 and 52) 3 and 73) 8 and 54) 8 and 7
[CATCATCAT]Mother 3[CATCATCATCATCATCATCATCAT]Mother 8
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Mother
3
Mother
8
Father
5 3,5 8,5Father
7 3,7 8,7
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How Do We Distinguish VNTR Alleles?
• Make DNA from tissue sample
• “Cut” DNA with Restriction Enzymes
• Separate resulting fragments by size with gel electrophoresis
• Transfer to filter
• Probe with complementary DNA that is “labeled”
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Restriction Enzymes
• Enzymes naturally found in bacteria, molds • Enzymes “cut” specific DNA sequences (sequence
of bases), yielding DNA fragments of various lengths
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Template
Complement
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Restriction enzyme cuts produce DNA fragments of various lengths
2.5 kb 15 kb 8.4 kb
3.7
kb
1.2
kb
2.3
kb
Section of chromosome
Sites where restriction enzyme cuts DNA
Polymorphic sites; some individuals have this site, some don’t
Copyright 2002 Prentice-Hall
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Gel Electrophoresis
• Use of electric current to separate DNA fragments of various lengths in an agarose gel
• Analogy: Long and short spaghetti through a colander
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Sample 1Samples
Double-strandedDNA
1. Restriction enzyme cuts DNA into fragments of various length.
Double-strandedDNA
2. The DNA sample is loaded onto a gel for electrophoresis.
3. Electrophoresis separates DNA fragments by charge and size. Small fragments run faster.
Restriction Digest and Electrophoresis
Copyright 2002 Prentice-Hall
+
-
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Stack ofblotting paper
Filter
Gel
Sponge inalkaline solution
Stack ofblotting paper
Filter
Gel
Sponge inalkaline solution
DNA probe in solution in plastic bag
Stack ofblotting paper
Filter Gel
Sponge inalkaline solution
DNA probe in solution in plastic bag X-ray film
4. The DNA fragments are treated with an alkaline solution to make them single stranded.
Single-strandedDNA
5. Blotting. An alkaline solution wicks up into blotting paper, carrying DNA from gel onto nylon filter, where it becomes permanently bound.
6. Hybridization with radioactive probe. Incubate the nylon membrane with a solution containing labeled probe DNA. The radioactive probe base pairs to the fragments containing complementary sequences.
7. Autoradiography. Place membrane against X-ray film. Radioactive DNA fragments expose film, forming black bands that indicate location of target DNA.
1 2 3 4
Copyright 2002 Prentice-Hall
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5’ CATCATCAT 3’
3’ GTAGTAGTA 5’
Template
Probe (complement)
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Uses of DNA Profiling• Testing Twins in utero to Determine if They are
Identical
• Identification of Bone Marrow Transplant Sucess
• Tumor Detection and Identification of Metastasis
• Identification of Pathogens
• Paternity Testing
• Diagnosis of Genetic Disease
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Both polymorphic sites present
Left present, right absent
Both polymorphic sites absent
Left absent, right present
17.5 kb 15.0 kb
8.4 kb
4.9 kb
3.7 kb
1.2 kb
2.5 kb 2.3 kb
17.5 kb 15.0 kb
8.4 kb
4.9 kb
3.7 kb
1.2 kb
2.5 kb 2.3 kb
A B C D
Copyright 2002 Prentice-Hall
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5’ CATCATCAT 3’
3’ GTAGTAGTA 5’
TemplateStrand
ComplementaryStrand
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HOW UNIQUE NUMBERS OF SIMPLE SEQUENCE REPEATS ARE GENERATED
1 2 3 4 6 75 8
1 2 3 4 6 75 8
1 2 3 4 6 75 8
1 2 3 4 6 75 8
1 2 3 4 6 75 865
1 2 3 4 7 8
1. Start with twochromosomeselections containingthe same simplesequence repeats.
2. The repeatsmisalign duringmeiosis l. Crossingover andrecombinationoccurs.
3. Meiotic productshave unique numberof repeats.
8repeats
8repeats
10repeats
6repeats
Copyright 2002 Prentice-Hall
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