1. HOW MANY CATs?
A DNA Profiling Simulation

2. 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. 5’ 3’ 3’ 5’

6. 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 ( 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]

7. 23 chromosomes from each parent = 46 total chromosomes in each child

8. [CATCATCAT]
Mother 3
[CATCATCATCATCATCATCATCAT] 8
[CATCATCATCATCAT]
Father 5
[CATCATCATCATCATCATCAT] 7
Child’s possible VNTR alleles at this locus on chromosome 17:
1) 3 and 5
2) 3 and 7
3) 8 and 5
4) 8 and 7

9. Mother 3 8
Father 5
3,5
8,5 7
3,7
8,7

10. 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”

11. Restriction Enzymes Enzymes naturally found in bacteria, molds
Enzymes “cut” specific DNA sequences (sequence of bases), yielding DNA fragments of various lengths

12. Template
Complement

13. Restriction enzyme cuts produce DNA fragments of various lengths
Sites where restriction enzyme cuts DNA
Section of chromosome
Polymorphic sites; some individuals have this site, some don’t
2.5 kb
15 kb
8.4 kb
Figure: 17.6a
Caption:
Genetic markers identify certain locations in the genome and vary among individuals. This diagram shows how the fragments of DNA generated by restriction enzyme cuts can be used as genetic markers.
3.7 kb
1.2 kb
2.3 kb
Copyright 2002 Prentice-Hall

14. 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

15. - + Restriction Digest and Electrophoresis Samples Sample 1
Double- stranded DNA
Sample 1
Restriction Digest and Electrophoresis -
Double- stranded DNA +
Box 15.2, Figure 1, steps 1-3
Caption:
Question  The intensity of a band in an autoradiograph indicates how much target DNA was in the sample. The darker the band, the more DNA. Why?
3. Electrophoresis separates DNA fragments by charge and size. Small fragments run faster.
1. Restriction enzyme cuts DNA into fragments of various length.
2. The DNA sample is loaded onto a gel for electrophoresis.
Copyright 2002 Prentice-Hall

18. Stack of blotting paper
Filter
Gel
Sponge in alkaline solution
DNA probe in solution in plastic bag
X-ray film
Stack of blotting paper
Filter
Gel
Sponge in alkaline solution
DNA probe in solution in plastic bag
Stack of blotting paper
Filter
Gel
Sponge in alkaline solution
Single- stranded DNA
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.
4. The DNA fragments are treated with an alkaline solution to make them single stranded.
5. Blotting. An alkaline solution wicks up into blotting paper, carrying DNA from gel onto nylon filter, where it becomes permanently bound.
7. Autoradiography. Place membrane against X-ray film. Radioactive DNA fragments expose film, forming black bands that indicate location of target DNA.
Box 15.2, Figure 1, steps 4-7
Caption:
Question  The intensity of a band in an autoradiograph indicates how much target DNA was in the sample. The darker the band, the more DNA. Why?
Copyright 2002 Prentice-Hall

19. 5’ CATCATCAT 3’ 3’ GTAGTAGTA 5’ Template Probe (complement)
----- Meeting Notes (7/12/11 11:23) -----
use of the word allele?

21. 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

23. 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
Both polymorphic sites present
Left present, right absent
4.9 kb
4.9 kb
Both polymorphic sites absent
3.7 kb
3.7 kb
Left absent, right present
2.5 kb 2.3 kb
2.5 kb 2.3 kb
Figure: 17.6b
Caption:
The banding patterns in part (b) are those predicted for individuals who are homozygous for each pattern.
1.2 kb
1.2 kb
Copyright 2002 Prentice-Hall

25. 5’ CATCATCAT 3’
3’ GTAGTAGTA 5’
Template
Strand
Complementary
Strand

27. HOW UNIQUE NUMBERS OF SIMPLE SEQUENCE REPEATS ARE GENERATED
1. Start with two
chromosome
selections containing
the same simple
sequence repeats. 1 2 3 4 5 6 7 8 8
repeats 8
repeats 1 2 3 4 5 6 7 8 1 2 3 4
2. The repeats
misalign during
meiosis l. Crossing
over and
recombination
occurs. 5 6 7 8 3 4 5 6 1 2 7 8
Figure: 16.8
Caption:
Because simple sequence repeats are so similar, they are likely to misalign when homologous chromosomes synapse at meiosis I. 1 2 3 4 5 6 5 6 7 8 10
repeats
3. Meiotic products
have unique number
of repeats. 1 2 3 4 7 8 6
repeats
Copyright 2002 Prentice-Hall