Loading... Please wait...

DRD4 Genotyping

Dopamine receptor D4 (DRD4) is a signaling protein in humans that is activated by the neurotransmitter dopamine. Mutation to DRD4 have shown to be associated with risk taking behaviour and many other things. Specifically the mutation is a 48 base pair(bp) Variable Number Tandem Repeat(VNTR) of DNA that is copied 2-11 times in the gene, increasing its size from 361 bp to over 600 bp. One of the most common versions of the mutation is the 7 Repeat or 7R of the 48 bp region.

 

Protocol

 

  1. Rinse your mouth with water and then rub inside of cheek with tip of cotton swab

  2. Take cheek swab and place in 300uL 50mM NaOH in microcentrifuge tube

  3. Stir swab in tube for 1 minute until solution is cloudy

  4. Heat tube in almost boiling water for 10 minutes (95C)

  5. Add 300uL of 50mM Tris

  6. For a 50uL PCR reaction

    1. 1uL of cheek cell solution for PCR reaction template

    2. 1uL of DRD4 primer

    3. 10uL of 5x Master Mix(or 25uL of 2x Master Mix)

    4. 38uL water with 5x Master Mix(or 23uL with 2x Master Mix)

  7. See PCR temperatures and times below

  8. First, make TAE if you don’t have it by adding 5g of TAE mix to 1L of distilled water

  9. Next, add 0.5g of agarose to 50mL of TAE(DON’T USE WATER!) and microwave it on high for 1 minute or until all agarose is dissolved(the solution should be clear)(How to cast an agarose gel)

  10. Add 5uL of DNA Dye to the still hot molten agarose solution and swish around to mix

  11. Dump the molten agarose into your gel mold and let set (this usually takes around 20-30 minutes but can be sped up by placing it in the fridge)

  12. Add 4-5uL of 100bp DNA ladder to the first or last lane of the gel

  13. Add 9uL of DNA loading buffer to your PCR reaction and then add 20uL to the gel






  • Use the PCR protocol below

 

1 x 95C 10 Minutes

 

30 x

95C 15s

55C 1 minute

72C 2 minute

 

1x 72C 10 minutes





Results

Compare the samples to the DNA ladder using a transilluminator  Individuals with genes around 300-400bp have a wild type gene(normal human gene) and those who have 600+bp have a version of DRD4 that is associated with risk taking behaviour!