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’

5’3’

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]

•23 chromosomes from each parent = 46 total chromosomes in each child

[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

Mother

3

Mother

8

Father

5 3,5 8,5Father

7 3,7 8,7

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

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

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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

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

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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

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5’ CATCATCAT 3’

3’ GTAGTAGTA 5’

Template

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

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

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5’ CATCATCAT 3’

3’ GTAGTAGTA 5’

TemplateStrand

ComplementaryStrand

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