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 2 minutes at maximum speed. This will cause a pellet of cells to form at the bottom of tube.

• Carefully pour off supernatant – DNA inside cells in pellet.  You should be able to see the pellet.  You will not be able to pour all the liquid out of the tube.

• Resuspend the cell pellet in the remaining saline solution and transfer 30 µL (with a P200) to an empty 200 µL PCR tube containing 100 µL 10% Chelex.

• When everyone is ready, place tubes into thermal cycler and incubate 10 minutes at 99 C. The heat will break open the cells, and the Chelex will remove Mg2+ and therefore stabilize the DNA.

• Place tubes into ice bucket until ready to add DNA to PCR Reaction tube.  Then centrifuge 5 minutes in minicentrifuge before adding 2.2 µL supernatant (containing DNA) to PCR reaction.

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

• Tube contains a dehydrated bead containing Taq DNA polymerase (enzyme) and dNTPs (building blocks)

• Add 20 mL primer mix containing both primers and buffer and dissolve bead

• Add 2.2 mL cheek cell DNA, Mix with pipet tip

• 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:

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

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

It is sequence-dependent:

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