HOW MANY CATs?
A DNA Profiling Simulation
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
5’
3’
3’
5’
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 allele
• Example: One allele might have 3 copies
[CATCATCAT] and the other allele might have 5 copies
[CATCATCATCATCAT]
•
•23 chromosomes from each parent = 46 total chromosomes
in each child
Mother
Mother
[CATCATCAT]
[CATCATCATCATCATCATCATCAT]
3
8
Father
Father
[CATCATCATCATCAT]
[CATCATCATCATCATCATCAT]
5
7
Child’s possible VNTR alleles at this locus on chromosome 17:
1)
2)
3)
4)
3 and 5
3 and 7
8 and 5
8 and 7
Mother
Mother
3
8
Father
5
3,5
8,5
3,7
8,7
Father
7
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”
Restriction Enzymes
• Enzymes naturally found in bacteria, molds
• Enzymes “cut” specific DNA sequences (sequence
of bases), yielding DNA fragments of various
lengths
Template
Complement
Restriction enzyme cuts produce DNA fragments of various lengths
Sites where
restriction enzyme
cuts DNA
Section of
chromosome
2.5 kb
Copyright 2002 Prentice-Hall
15 kb
8.4 kb
Polymorphic sites;
some individuals
have this site,
some don’t
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
Restriction Digest and Electrophoresis
Samples
Sample 1
Doublestranded
DNA
1. Restriction enzyme cuts
DNA into fragments of
various length.
Copyright 2002 Prentice-Hall
Doublestranded
DNA
+
2. The DNA sample is
loaded onto a gel for
electrophoresis.
3. Electrophoresis
separates DNA fragments
by charge and size. Small
fragments run faster.
DNA probe
probe in
in
SingleDNA
solution in
stranded
solution
in
plastic bag
1 2 3 4 DNA
plastic bag
Stack
of
Stack
of
blotting paper
blotting paper
Filter
Filter
Gel
Gel
Sponge in
in
Sponge
alkaline solution
alkaline solution
6. Hybridization with
4. The DNA
5. Blotting. An
radioactive probe.
fragments are alkaline solution
Incubate the nylon
treated with an wicks up into
membrane with a solution
alkaline
blotting paper,
containing labeled probe
solution to
carrying DNA from
DNA. The radioactive
make them
gel onto nylon filter, probe base pairs to the
single
where it becomes
fragments containing
stranded.
permanently bound. complementary
sequences.
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X-ray film
7. Autoradiography.
Place membrane
against X-ray film.
Radioactive DNA
fragments expose film,
forming black bands
that indicate location of
target DNA.
5’ CATCATCAT 3’
Template
3’ GTAGTAGTA 5’
Probe
(complement)
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
A
B
C
D
17.5 kb
15.0 kb
17.5 kb
15.0 kb
8.4 kb
8.4 kb
4.9 kb
4.9 kb
3.7 kb
3.7 kb
2.5 kb
2.3 kb
2.5 kb
2.3 kb
1.2 kb
1.2 kb
Copyright 2002 Prentice-Hall
Both polymorphic sites present
Left present, right absent
Both polymorphic sites absent
Left absent, right present
5’ CATCATCAT 3’
Template
Strand
3’ GTAGTAGTA 5’
Complementary
Strand
HOW UNIQUE NUMBERS OF SIMPLE SEQUENCE REPEATS
ARE GENERATED
1
2
1
2
1
2
3
4
5
6
7
8
3
4
5
6
7
8
3
4
5
6
7
8
6
1. Start with two
chromosome
selections containing
the same simple
sequence repeats.
8
repeats
8
repeats
1
2
1
2
3
4
5
3
4
5
6
5
10
repeats
1
6
repeats
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2
3
4
7
8
7
6
2. The repeats
misalign during
meiosis l. Crossing
over and
recombination
occurs.
8
7
8
3. Meiotic products
have unique number
of repeats.