HOW MANY CATs? A DNA Profiling Simulation

Background Information 1. Structure of DNA

• Double helix

• Anti-parallel strands

• 4 Bases (A, C, G, and T)

• Complementary bases

A A T T

G C

A A T T

G C

Template Strand

Complementary Strand

5’ 3’

5’ 3’

Background Information 1. Structure of DNA

•Double helix

•Anti-parallel strands

•4 Bases (A, C, G, and T)

•Complementary bases pair

•Negatively charged molecule

•Organized into chromosomes

A A T T

G C

A A T T

G C

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Gametogenesis

Background Information 2. Inheritance of Chromosomes

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•We have two pairs of chromosomes and two copies (alleles) of each gene.

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

•During meiosis I, crossing-over and recombination may occur between the homologous chromosomes, resulting in rearrangement of the DNA.

replication of starting chromosomes

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Background Information 3. Variable Number Tandem Repeats (VNTRs)

• “Junk” DNA that likely does not code for any protein

• Short sequences (3-30 bp) that are repeated multiple times (10-100 times) Example: CATCATCATCAT…

5’ C AT C AT C AT 3’

3’ GTA GTA GTA 5’

Template strand

Complementary strand

Background Information 3. Variable Number Tandem Repeats (VNTRs)

• 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]

[CATCATCAT] Allele 1 3 [CATCATCATCATCATCATCATCAT] Allele 2 8

Mother

[CATCATCATCATCAT] Allele 1 5

[CATCATCATCATCATCATCAT] Allele 2 7 Father

Child’s possible VNTR alleles at this locus on chromosome 17:

3 and 5

3 and 7

8 and 5

8 and 7

How are unique numbers of simple sequence repeats generated?

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Start with two chromosome selections containing the same simple sequence repeats.

1 2 3 4 6 7 5 8 8 repeats

8 repeats

1 2 3 4 6 7 5 8

1 2 3 4 6 7 5 8

1 2 3 4 6 7 5 8

The repeats misalign during meiosis I. Crossing over and recombination occur.

Meiotic products have a unique number of repeats.

1 2 3 4 6 7 5 8 6 5

1 2 3 4 7 8

10 repeats

6 repeats

Background Information 4. Restriction Enzymes

• Naturally found in bacteria

• “Cut” specific DNA sequences (sequence of bases), yielding DNA fragments of various lengths

+

-

Background Information 5. Gel Electrophoresis

• Because DNA is negatively charged, when it is loaded into an agarose gel and subjected to an electric current, it will move away from the anode and toward the cathode.

• This allows separation of DNA fragments based on length, as smaller DNA fragments move more quickly through the gel.

+

-

Background Information 5. Gel Electrophoresis

• Because DNA is negatively charged, when it is loaded into an agarose gel and subjected to an electric current, it will move away from the anode and toward the cathode.

• This allows separation of DNA fragments based on length, as smaller DNA fragments move more quickly through the gel.

6. Southern Blotting

• After transfer to a nylon membrane, DNA fragments from the gel are “probed” with complementary sequence fragments that are labeled, allowing visualization of the target DNA bands.

DNA is invisible in the gel until it is stained or probed.

If the gel is stained with a DNA dye, all DNA in each well becomes visible – as a smear.

5’ C AT C AT C AT 3’ Target DNA (in template strand)

DNA probes are composed of sequences which complement the target sequence and can be labeled with radioactive or fluorescent tags.

Probe (complement) 3’ GTA GTA GTA 5’

If we use a specific probe to find only fragments of interest, meaningful bands emerge.

Restriction Digest & Electrophoresis

Double- stranded DNA

Restriction digest. Restriction enzyme cuts DNA into fragments of various lengths.

1

Single- stranded DNA

Denaturation. The DNA fragments are treated with an alkaline solution to make them single stranded.

3

Gel electrophoresis. DNA fragments are separated by charge and size. Small fragments run faster.

2

Double- stranded DNA

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Southern Blotting

Blotting. An alkaline solution wicks up into blotting paper, carrying DNA from gel onto nylon membrane, where it becomes permanently bound.

Stack of blotting paper

Membrane

Gel

Sponge in alkaline solution

4 DNA probe in solution in plastic bag

Hybridization with a radioactive OR fluorescent probe. The nylon membrane is incubated with a solution containing labeled probe DNA. The probe base pairs to the fragments containing complementary sequences.

5

X-ray film Autoradiography. The membrane is placed against X-ray film. Radioactive DNA fragments expose film, forming black bands that indicate location of target DNA.

6 Fluorescent visualization. Under the correct wavelength of light, the probe will fluoresce, allowing the bands where it has bound to target DNA to be observed.

OR

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HOW MANY CATs?

1 Restriction digest Cut the DNA samples at restriction sites.

2-4 Gel electrophoresis, denaturation, and blotting

Arrange DNA fragments and ladder on “gel” paper.

5 Probe hybridization Match probes to CATCAT sequences on the gel.

6 Visualization Analyze probe-labeled bands for genotypes.

Protocol Simulation

Keep in Mind…

1. Your “DNA samples” show only 1 strand of DNA for each allele (2 alleles per subject) – no complementary strand.

2. It’s a good idea to mark your restriction sites before cutting – check twice, cut once.

3. DNA is negatively charged, so on your “gel” it will from the anode (-) to the cathode (+). Be sure to mark your anode and cathode on your gel.

4. Larger DNA takes longer to make its way through the gel, so smaller fragments will be furthest from the anode. Make sure you spread out your fragments in the right direction.

5. On a real gel, the DNA fragments would be invisible in the gel until they are labeled with the fluorescent probe. That’s why the probing step is so important!