Lab Protocol for DNA Isolation and Polymerase Chain Reaction

This protocol involves isolating your own DNA from cheek cells and conducting a genotype analysis. We will analyze the PTC gene which contributes to our ability to taste a bitter compound called PTC. Neither of the two phenotypes (taster or nontaster) is advantageous over the other. Please let your instructor know if you have any concerns about participating.

DNA isolation Procedure

1. Each student will take a small paper cup containing 10 mL 0.9% NaCl saline which is isotonic to cells.
2. Vigorously rinse mouth with 10 mL 0.9% Saline solution for 30 seconds and spit back into cup.
3. Swirl cup to mix cells and immediately transfer 1 mL of solution with P1000 to Eppendorf tube.
4. When everyone is ready, place tubes into centrifuge in a balanced configuration and centrifuge for 60 seconds at maximum speed. This will cause a pellet of cells to form at the bottom of tube.
5. Carefully pour off supernatant – this is the liquid portion of your tube. The DNA inside cells in pellet at the bottom of the tube.  You should be able to see the pellet.  You will not be able to pour all the liquid out of the tube.
6. Resuspend the remaining cells in the tube by using the vortex to shake the tube and place on ice until you are ready to set up the PCR tubes.

The Polymerase Chain Reaction

• A thermal cycler is needed to repeatedly change the incubation temperature

• 94 C for 30 sec: to separate double-stranded DNA into single strands

• 54 C for 30 sec: to allow primers to hybridize (anneal) to DNA templates

• 72 C for 30 sec: optimum temperature for function of Taq DNA polymerase

• We will use 35 cycles of the three temperature steps indicated above

The required ingredients in test tube:

• Template DNA: pre-existing DNA molecules to be copied – isolated from cheek cells

• Taq DNA polymerase: enzyme that remains functional at high temperature – in freeze-dried pellet present in PCR tube.

• dNTPs: monomers for the synthesis of new DNA strands present in freeze-dried pellet

• Many copies of 2 different primers: one to hybridize to each strand of double-stranded DNA. Primers determine where DNA synthesis will begin – in primer mix

• Buffer: ensures proper pH and provides Mg2+ cofactor – in primer mix

Set up of PCR Reaction

1. The PCR tubes are attached to each other. It is best to leave them attached and label each one with a mark to distinguish each group member’s tube.
2. Each student will add:
   1. 1. 20 µL of primer mix to their tube
      2. 2.2 µL of their resuspended cells (make sure to mix by pipetting or vortexing before measuring)

3. Students may quickly centrifuge their PCR tube for 2-3 seconds before placing in the thermal cycler to ensure all the liquid is settled and no air bubbles exist. When everyone is ready, quickly place tubes into thermal cycler under the guidance of your instructor. The instructor will provide a diagram to ensure a map of where each group has place their tubes.

4. Once the PCR protocol is complete, the instructor will gather tubes with amplified DNA and place in the freezer for next week.

Primer Annealing Activity

While the PCR protocol takes about 1 hour, students will complete their Student Data Sheet to work on gaining clearer understanding of the Polymerase Chair Reaction.

Primer annealing is temperature dependent:

• More hydrogen bonds connecting primer to template means higher temperature necessary to break the interaction

• Scientist controls hybridization (annealing) temperature of thermal cycler such that a near perfect match is necessary for binding

It is sequence-dependent:

• Scientist chooses the sequences of the primers used in the PCR reaction according to DNA segment to be amplified