15 Lab Protocol: DNA and PCR

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

• Everyone will take a 50 mL conical tube containing 10 mL 0.9% Saline (isotonic).

• Vigorously rinse mouth with 10 mL 0.9% Saline solution for 30 seconds and spit back into tube.

• Swirl tube to mix cells and immediately transfer 1 mL of solution with P1000 to Eppendorf tube.

• When everyone is ready, place tubes into centrifuge in a balanced configuration and centrifuge for 90 seconds at maximum speed. This will cause a pellet of cells to form at the bottom of tube.

• 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.

• Add 22.5 uL of PTC primer/loading dye mix to the Ready to Go PCR bead

• Add 2.5 uL of the cell solution that you just resuspended.

• Follow instrucions for setting up the Polymerase Chain Reaction below.

The Polymerase Chain Reaction

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

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

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

• 72o 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

• Assemble ingredients into PCR tube on ice

• Label a PCR tube with ID number

• When everyone is ready, quickly place tubes (have limited time frame) into PCR machine with the following program:

• 94o C 2 minutes

• 94o C 30 seconds, 54o C 30 seconds, 72o C 30 seconds = 1 cycle, 35 cycles

• 4o C to refrigerate forever

• Instructor will place tubes in freezer until ready to analyze results in two weeks

Primer Annealing Activity

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