1. ALL THAT COMES
WITH GENOTYPING
Department of Environmental and Molecular Toxicology
Presenter: Lubna Khan
Faculty Advisor: Nancy Kerkvliet, PhD

2. Modern Medicine
 Specific genes associated with certain
diseases
 Focus on Immune System related diseases

3. Type 1 Diabetes
 In type 1 diabetes,
the body does not
produce insulin.
 The immune system
mistakenly sees the
insulin-producing
cells (Beta-cells) in
the pancreas as
foreign, and
destroys them. This
attack is known as
"autoimmune"
disease.

4. Welcome to Kerkvliet Lab:
Ah Receptor
What genes suppress this autoimmune
response?

5. Research Question
Q. Does the lack of
AHR alter the
progression of Type 1
diabetes?

6. NOD Mouse Model
 Non-Obese Diabetic (NOD) Mice Spontaneously
develop T1D (90-100% by 30 weeks of age)
 Generate NOD Mice with no AhR
 AhR KO Mice

7. Breeding
 Wild type (Wt):
 AhR +/+
 Heterozygous (Het):
 AhR +/-
 Mutant (Mut):
 AhR -/-

8. Summer Research Question
Q. Which mice lack
AhR?

9. Genotyping
 The process of determining the genetic make-
up of an organism.
 DNA Extraction
 Polymerase Chain Reaction (for genes of
interest)
 Amplification of AhR gene
 Gel Electrophoresis

10. DNA Extraction
 Tail snips
 Tail Digestion
 DNA Extraction

11. Polymerase Chain Reaction
(PCR)

12. Gel Electrophoresis
 A method for separation
and analysis of DNA
fragments based on
size.
 Wt fragment: 670 BP
 Mut fragment: 450 BP
 Het: Both 670 BP and
450 BP fragment

13. Expected Result

14. NOD Mice Genotyping Attempt#1

15. And #2 and #3..
Ladder showed up, but no bands:Partial ladder?

16. The Protocol
 Pipetting (?)
 DNA Extraction
 PCR Reaction
 0.5µL DNA
 6.9µL dH20
 1µL 10X Buffer
 0.2µL DNTPs (?)
 0.4µL JAX MUT
 0.4µL JAX WT
 0.4µL JAX CMN
 0.2µL Taq
 Gel

17. The Protocol
 Pipetting (?)
 DNA Extraction ✓
 PCR Reaction
 0.5µL DNA ✓
 6.9µL dH20 ✓
 1µL 10X Buffer ✓
 0.2µL DNTPs (?)
 0.4µL JAX MUT
 0.4µL JAX WT
 0.4µL JAX CMN
 0.2µL Taq
 Gel

18. Troubleshooting
New DNTPs with controls:
Better, but missing a
band on het
Old DNTPs with controls:

19. The Protocol
 Pipetting (?)
 DNA Extraction ✓
 PCR Reaction
 0.5µL DNA ✓
 6.9µL dH20 ✓
 1µL 10X Buffer ✓
 0.2µL DNTPs ✓
 0.4µL JAX MUT (?)
 0.4µL JAX WT (?)
 0.4µL JAX CMN (?)
 0.2µL Taq
 Gel

20. Primers
Three Primers Separated Primers

21. Separate the Primers

22. The Protocol
 Pipetting (?)
 DNA Extraction ✓
 PCR Reaction
 0.5µL DNA ✓
 6.9µL dH20 ✓
 1µL 10X Buffer ✓
 0.2µL DNTPs ✓
 0.4µL JAX MUT ✓
 0.4µL JAX WT ✓
 0.4µL JAX CMN ✓
 0.2µL Taq
 Gel ✓

23. The NOD Finale

24. Research Question Answered

Which mice lack AHR?
 Mice were genotyped and the AhR KO mice were
identified.

25. Next Steps
 How does AHR deficiency alter the
progression of Type 1 diabetes?
 Monitor Blood Glucose Levels (BGLs) and weight
 Use wild type mice for comparison of disease
progression

26. Acknowledgements
 Kerkvliet Lab
 Nancy Kerkvliet
 Jamie Pennington
 Sumit Punj
 Kevin Ahern