The Great Kennel Conundrum
Introduction: Hybrid or designer dogs have become a very popular to domestic animal owners
since the 20th century. The idea is two cross two pure bred dogs to produce a mix or hybrid. This is
a form of selective breeding. An example is crossing between a Pug and a Poodle (two purebreds)
to form a Pugapoo. The list is long and can get quite capricious. There are Basscotties,
Carnauzers, Cockapoos, Frengles, Gollies, Pug-zus, Rat-a-paps, Schneagles, and Schnoodles.
Although designer dogs are often celebrated for their novelty, an underlying motive for
hybridization is an attempt to reduce the incidence of certain hereditary problems found in purebred
dogs. It is possible to visualize the how the hybrids relate to the two pure breds through DNA
analysis.
DNA is the genetic biochemical compound found in the nucleus of eukaryotic cells. In
humans, 99.9% of DNA is the same in every person. But it is the 0.01% that accounts for the vast
differences between each person. Only identical twins have 100% identical DNA. The dog genome
is very similar to humans but divided up in greater number of chromosomes (46 in humans, 78 in
canines) but the same technology can be used to identify one dog from another.
DNA experts can utilize two main technologies to DNA. One is called Polymerase Chain
Reaction or PCR which makes millions of copies of selected DNA sequences that can be analyzed
using Gel Electrophoresis. Another technique is Restriction Fragment Length Polymorphism or
RFLP. In RFLP, restriction enzymes are used to cut DNA sequences are specific sites creating
fragments. These fragments are also analyzed using Gel Electrophoresis which uses an electrical
current in an agarose gel to separate the DNA fragments according to size.
Restriction enzymes are found in all bacteria as a defense mechanism against viral DNA.
Scientists have isolated over 3000 different restriction enzymes from different strains of bacteria.
Some restriction enzymes such as HindIII, EcoR1, and BamH1 cut DNA sequences at very specific
sites. In the RFLP technique which is also called DNA fingerprinting, a restriction enzyme is added
to a purified DNA sample. Then the DNA is loaded in an agarose gel. When connected to an
electrical field, the DNA is pulled to the positive end of the gel. The smaller DNA fragments are
pulled through the gel faster than the larger ones. When stained, a DNA banding pattern creates the
fingerprint.
Scenario: The Shane family bought their hybrid dog, a Labradoodle named Shelby from Pipton
Kennels. A year earlier the Shane’s had visited the kennel and picked out the sire (male), a brown
haired Labrador retriever and a dame (female) a white French Poodle. But when they showed
Shelby to another canine breeder he questioned the dog’s ancestry Was Shelby the right hybrid?
The breeder offered to have Shelby’s DNA analyzed to determine if it really was a Labradoodle.
Labradoodle
Labrador retriever
Poodle
Objective: To determine the ancestral identity of a hybrid dog by the use of DNA technologies.
Task 1: Simulate the action of restriction enzymes
DNA scientists use restriction enzymes to cut DNA at specific sites. One of the first enzymes
identified was from E.coli and is named EcoR1. The enzyme EcoR1 will look for a specific
palindrome sequence:
What’s a palindrome? ____________________________________________________.
So look at the double stranded sequence:
CTGAAGAATTCGAAGGTTT
G A C T T C T T A A G C T T C C A A A.
EcoR1 will scan it looking or GAATTC.
Do you see the sequence?
When it find the sequence it makes a cut in between the G and A; and then through the hydrogen
bonds to create a cut called a “sticky end”. They are sticky because they can recombine.
CTGAAG AATTCGAAGGTTT
G A C T T C T T A A G C T T C C A A A.
CTGAAG
GACTTCTTAA
AATTCGAAGGTTT
G C T T C C A A A.
The result is two fragments are
produced
Now you find the sites:
CTGAAGAATTCGAAGGTTTCTGAAGAATTCGAAGGTTT
G A C T T C T T A A G C T T C C A A A.G A C T T C T T A A G C T T C C A A A
How many restriction sites are there? _____ How many fragments are produced? ______
Observe the restriction sites and the number and sizes of the fragments.
4 kb
3.5 kb
2.5 kb
5.0 kb
Dog 1DNA
6 kb
4 kb
3 kb
2 kb
Dog 2 DNA
8 kb
Dog 3 DNA
4.5
kb
2.5 kb
Name______________________________________________Period ____Due Date_________
Turn in this sheet only
Task 2: Simulate gel electrophoresis by completing the gel outline on the agarose gel.
Remember the shorter the DNA fragment, the longer it travels in the gel.
Dog 1 has been done for you.
Agarose Gel Outline
Dog 1
Dog 2
Dog 3
Wells
Fragment
Size
8 kb
7 kb
6 kb
5 kb
4kb
3 kb
2 kb
1kb
1. In the gel outline above, which restriction digest produced the largest Dog DNA fragment?
___________________.
2. In the gel outline above, which restriction digest produced the shortest Dog DNA fragment?
___________________.
3. Aside from identifying hybrid dogs, name three societal uses for DNA Fingerprinting.
_______________________, ____________________, _________________________
Task 3: DNA Fingerprinting (Wet-lab)
Procedures:
1. Once your agarose gel has been formed in the gel chamber and has been covered with TBE
buffer, set the p20 micropippettor to 10 ul and load into the designated well. (see gel image below).
Suspect 3
Suspect 2
Suspect 1
Evidence
Evidence
Suspect 1
Suspect 2
Suspect 3
2. Make sure you change tips before loading each sample.
3. When all DNA samples have been loaded, cover the gel box and connect the leads.
4. Gels will run (electrical current) for 20-30 minutes at 100 volts.
5. After staining with methylene blue stain, the gels may be analyzed 24 hours later.
Data: Copy the banding pattern of your agarose gel in the diagram below left.
Evidence
Suspect 1
Suspect 2
Suspect 3
Legend
1
2
3
1 – DNA pattern from
a Labradoodle
2 – Golden doodle
3 – Cockadoodle
Questions:
1. What hybrid dog did the Shane’s get? _______________________________________.
2. What is the purpose of TBE Buffer? _________________________________________
______________________________________________________________________.
3. Why do the DNA fragments migrate through the agarose gel? _____________________
______________________________________________________________________
4. Why is loading dye added to the DNA samples? (2 reasons) _______________________
_______________________________________________________________________