Modeling PCR

1. Modeling PCR Joy Killough RET Teacher University of Texas at Austin With Dr. C. Randall Linder Environmental Science Institute Dr. Jay Banner, Director A Research Experience for Teachers Lesson Program Funding from National Science Foundation

2. Modeling PCR Techniques in molecular biology are sometimes difficult for students to grasp since events in the test tube cannot be seen. This activity models the process of PCR, polymerase chain reaction. PCR is a very common, heavily used procedure in the molecular biology lab since it allows a tiny amount of DNA to be amplified more than a million times in just a few hours. The use of transparency film eliminates the time consuming cutting of nucleotides models. A modest investment in a box of ink-jet transparency film allows a teacher to print the templates in color from a desk top printer. Models are reusable. The models may be printed directly from the PowerPoint by choosing “print” then “slide.”

3. Materials Needed For each student group provide • Transparency Sheet “Modeling PCR Reaction Chamber” • Forward and reverse primers (cut apart into individual primer strips of six nucleotides each). DNA strands cannot begin from “scratch” but require a starter piece or “primer.” A primer is required for both strands of the DNA being copied therefore both forward and reverse primers are needed. • Template DNA. This is the DNA chosen for replication. • Temperature strip (to simulate the cyclic temperature changes in the thermocycler) • 1” wide strips of blank transparency film (to build new DNA strands on and allow subsequent repositioning) • Tape (to keep primers in position on transparency strip) • ¼” color dots from the office supply store, assorted color pack (red, yellow, blue, green). The dots model dNTP’s, the nucleotide source for the new strands. • Scissors if pieces are not already cut apart.

4. Procedure • Set up model • Place “Modeling PCR” transparency on a light colored surface. Cut out the temperature strip and slide under the far right section labeled “temperature” so that the first temperature in the cycle (94°, denaturing) appears in the window. • Cut apart and place both forward and reverse primers in the primer section at the top left. • Place template DNA in the center of the page. • Place small colored dots representing dNTP’s in the upper right where labeled. • Discuss model pieces as they are placed. • The PCR reaction chamber models what occurs inside of a small test tube placed in a thermocycler. This piece of equipment, as the name implies, cycles through a temperature sequence programmed in by the operator. The sequence is commonly repeated 28-29 times in one PCR. • Temperature strip- A high temperature is used to denature the DNA, separating it through the hydrogen bonds. This is followed by a lower temperature where the primers “anneal” or base pair with the DNA. Next, a slight temperature rise favors the addition of dNTP’s during the extension phase. • Primers- excess primers are added to decrease the likelihood of denatured DNA base pairing with other denatured DNA. DNA requires a primer to begin replication. • Template DNA- this is the DNA the researcher wants multiple copies of. It may be anything from a piece of bacterial DNA being studied to DNA from a defendant’s blood sample. • dNTP’s- each colored circles represents a nucleotide. To represent base pairing match blue with green and red with yellow. The “n” in dNTP’s represents all four DNA bases. • Taq is not added as a model piece since it does not need manipulation but emphasize to students it is needed for PCR because it is a polymerase enzyme that does not denature at the high temperature needed for the denaturing of the DNA template.

5. Procedure Continued • Using the model. • If possible run the simulation on this PowerPoint to help clarify these instructions before using model with students. • The transparency model allows the teacher to use the overhead projector to help a large class place and manipulate pieces correctly. • With pieces in place and “94° denature” in the temperature window, separate the template DNA down the long axis where hydrogen bonds would be. I prefer to have the pieces already cut and show denaturing by pulling them apart. Place 2 narrow strips of transparency film beside the template DNA to build the new strands on. This allows them to be repositioned later. • Move the temperature strip up to the next setting (60° annealing). Match a forward and reverse primer by base pairing to the two 3’ ends of the template. You may tape these to the transparency strips to keep in place. • Move the temperature strip up to the next setting (72° extending). Add dNTP’s following the base pairing rules A with T (blue with green) and G with C (red with yellow). Extension should be from the primer so that the strand grows in a 5’ to 3’ direction. Continue until you reach the end of the template. • Repeat the cycle as many times as desired. You may combine student models at this point if you wish. • Evaluation- Questions are provided at the end if desired. This activity is also suitable for a performance based assessment with the student modeling the PCR on the overhead and explaining to the class what is occurring.

6. dNTP’s Modeling PCR Reaction Chamber TAQ Primers 94° Denature 60° Annealing 72° Extending Template DNA This process is taking place inside a small plastic microtube which is inside of a thermocycler.

7. Temperature Strip 94º Denaturing 60º Annealing 72º Extending 94º Denaturing 60º Annealing 72º Extending 94º Denaturing 60º Annealing 72º Extending Templates- Print one page per group on transparency film using your color printer. Alternately, make transparencies on the copy machine and add colored dots purchased from the office supply store, or color with permanent marker. A supply of colored dots (yellow, red, blue and green) are needed in any case. Template DNA- 3’ 5’ Primers Key: Phosphate Sugar Base Forward primer Reverse primer

8. Modeling PCR Reaction Chamber Temperature Nucleotides “dNTP’s” Primers (Forward and reverse) TAQ Add template DNA here This process is taking place inside a small plastic microtube which is inside of a thermocycler.

9. Modeling PCRStudent Questions • 1. What things are needed in a micro test tube to have a successful amplification? • 2. Outline the role of each of the items listed above. • 3. Give the reason for the three temperature shifts during the PCR cycles. • 4. What would happen if you failed to denature the DNA? • 5. What would happen if you did not add enough dNTP’s? • 6. What would happen if instead of using Taq you substituted a polymerase that denatured at 85°? • 7. What would happen if you only added forward primers? • 8. Research the discovery of the PCR process. • 9. Research the origin of Taq.

10. Forward primer Reverse primer Forward primer Forward primer Forward primer Reverse primer Reverse primer Reverse primer Extra Primers (Optional for Bulk Printing)

11. 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ Extra DNA Templates (Optional for Bulk Printing)

12. Answers • 1. What things are needed in a micro test tube to have a successful amplification? -Primers, dNTP’s, Taq polymerase, and template DNA. • 2. Outline the role of each of the items listed above. -Template DNA is the source of DNA to be copied. -Taq polymerase is an enzyme which does not denature at the high temperatures used to break the hydrogen bonds of DNA and which is used to add nucleotides. -dNTP’s are the nucleotide raw material. -Primers are short pieces of RNA which allow you to select the section of DNA you wish to copy.

13. Give the reason for the three temperature shifts during the PCR cycles. • The 94° step denatures the template DNA. At this temperature the hydrogen bonds between the strands breaks. • At 60°, annealing, the primer matches up with the sequence on the template DNA with which it base pairs. • At 72°, extending, the nucleotides are added to the primer based on base pairing rules with the template DNA strands. • What would happen if you did not add enough dNTP’s? • - At a certain point dNTP’s would be used up through incorporation in the growing DNA strands. At this point the strands would stop growing due to lack of building material. • What would happen if you failed to denature the DNA? • -The strands could not be replicated since the primer could not anneal and the dNTP’s could not base pair. • What would happen if instead of using Taq you substituted a polymerase that denatured at 85°? • - The DNA would not reach the temperature needed to denature before the polymerase denatured. Once the polymerase denatured it would be ineffective at building the DNA stands.

14. What would happen if you only added forward primers? • -Only one side of the DNA could copy. This would not allow for the logarithmic increase in the number of copies of DNA. • Research the discovery of the PCR process. • -Answers will vary. • Research the origin of Taq. • -Answers will vary

15. References • Campbell, Neil, and Jane Reece. Biology. 6th ed. San Francisco, California: Benjamin Cummings, 2002. • Raven, Peter, and George Johnson. Biology. 6th ed. Boston, Massachusetts: McGraw-Hill, 2002.