5.3: Part II- Paternity Case- Who is the Father of My Kittens?

Mary has a white cat named “Honey” who was lost for two days about three months ago. She now has four kittens (photo 1) and Mary wants to know if the two neighboring cats, “Tom” or “Butch,” could be the father of each kitten. To analyze their DNA fingerprint, Mary has collected hair follicles from each adult cat and kitten, extracted DNA, and amplified DNA using the polymerase chain reaction. 

Figure 11.2. Honey (left) and her four kittens
(in order): Cream, Molasses, Ginger, Sugar

 

Figure 11.3. Potential fathers: Tom (left) and Butch (right)

Hypothesis

Using the photo, complete table 1 with your prediction of the father of each kitten.

Table 11.1. Hypothesis on Paternity of Kittens

KITTEN	POTENTIAL FATHER	REASONING
Cream
Molasses
Ginger
Sugar

Materials 

Reagents 

• Pre-cast 0.8% agarose gel
• Seven samples in microfuge tubes. One for each feline
• 135-150 mL 1X sodium borate running buffer (enough to submerge the agarose gel)

Equipment and Supplies

• Electrophoresis system such as MiniOne or other brand with the gel chamber and power supply
• P-20 micropipettes and appropriate tips
• Cell phone or camera to photograph the gel to document results

Procedure

Note

The following is completed when the electrophoresis chamber has been prepared with an 0.8% agarose gel and 1x Sodium Borate buffer in the chamber. Do not forget to document your procedure appropriately in your laboratory notebook.

1. Obtain the “DNA samples” – there are seven microfuge tubes labeled P- V.
2. Using a P-20 micropipettor and a pipet tip, measure 10µL from Tube P and transfer into the first well of the agarose gel. Be sure to follow the gel loading order noted in Column 1 – Well.
3. Using a new tip for each sample, transfer 10µL of each sample into new wells of the gel.
4. Be sure to keep track of your sample loading, if you do not follow the table below. If there were any problems with the loading (punctured gel, not enough sample), be sure to write in the NOTE column. 

Loading notes: Include any deviations or notes in your laboratory notebook

Table 11.2. 

Well	Tube	DNA Sample 10µL	Notes for loading
1	P	Tom (male)
2	Q	Cream (kitten)
3	R	Molasses (kitten)
4	S	Honey (female)
5	T	Ginger (kitten)
6	U	Sugar (kitten)
7	V	Butch (male)

6. If using a MiniOne electrophoresis system, run the gel for 15 minutes, until color bands separate. If using another electrophoresis system, run the gel at 135V until the dye front is.
7. For best viewing of the results, pick up the casting tray (with gel) out of the buffer tank, slide the gel onto a white laminated paper, label the samples (and your team name) and take a photo. 
8. Because DNA samples will diffuse through the agarose gels, you should always record results quickly once the electrophoresis has been turned off.

Analysis

1. Use colored pencils to record the band patterns (color the appropriate blocks) in the Data Table below.

Table 11.3. Colored “DNA” Bands Separated by Agarose Gel Electrophoresis

Band	Tom Male	Cream	Molasses	Honey Female	Ginger	Sugar	Butch Male
Blue #1
Blue #2
Pink #1
Purple #1
Yellow #1
Yellow #2

2. Carefully consider each band of all four kitten samples and determine whether the band matches Tom, Honey or Butch. For the kitten samples (columns QRTU) in Data Table 3, write (within the colored blocks) who matches that band -- Tom, Honey, or Butch.
3. Draw your conclusions based on the “DNA evidence”. 
4. Fill in the 2nd and 3rd column in table 4 below. Compare your hypothesis in table 1 where you guessed the father for each kitten based on appearances to your conclusion regarding the father based on the DNA evidence. Was your hypothesis correct for each kitten?
5. What is the specific evidence that justifies your conclusion determining each kitten’s father? Fill your responses to these questions in the table below.

Table 11.4. Comparison of Hypothesis and Conclusion backed by Experimental Evidence

KITTEN	FATHER based on visual	FATHER based on DNA	Evidence
Cream
Molasses
Ginger
Sugar