PCR Fundamentals. Introduction

Transcription

1 PCR Fundamentals Introduction The Polymerase Chain Reaction (PCR) is arguably the most important technique in the molecular biologist s repertoire (important enough to win its inventors the Nobel Prize). In this exercise, you will become familiar with the technique and some of the parameters that affect its accuracy. Additionally, the entire class will participate in a community analysis study of the microbial community found in the termite hindgut by subsequently sequencing your PCR products generated in this week s exercise. Learning Objectives Conceptual To better understand the nature of the DNA molecule and its replication by its manipulation in the laboratory To understand that targeted regions of DNA sequences can be synthesized and amplified thousands of fold in vitro Practical To learn how to set up PCR reactions and the role of the various reaction ingredients. To learn the how varying reaction conditions can affect the efficiency and accuracy of the PCR reaction Underlying Science The Polymerase Chain Reaction (PCR) is an enzymatic method of synthesizing ( amplifying ) large quantities of a targeted region of DNA in vitro (extracellularly, in a test tube). Because you can potentially generate millions of copies of a specific segment of DNA--even from a single, initial copy--this technique has become a ubiquitous and powerful tool in diagnostics, forensics and research biology. Once amplified, PCR products can simply be visualized by agarose gel electrophoresis or can subsequently be digested with restriction enzymes for DNA fingerprinting, cloned or sequenced. PCR works by using a thermostable DNA polymerase and short DNA fragments, called primers, to direct the synthesis of a specifically-targeted segment of DNA. The synthesis is repeated numerous times, called cycles, allowing the products of previous synthesis cycles to serve as template for the next cycle, resulting in an exponential amplification of the targeted region of DNA. This repeated cycling is made possible by the use of Taq polymerase, a thermostable polymerase isolated from the thermophilic bacterium Thermus aquaticus, originally isolated from hot springs in Yellowstone National Park. Because all DNA polymerases require a short segment of double-stranded DNA to begin DNA synthesis, short,complementary, oligonucleotides called primers are used to initiate DNA synthesis at the defined target region. The primers (also called oligonucleotides meaning short nucleotides) are synthesized chemically and can be designed to be complementary to any known sequence of DNA. They can range in size from 10 to 100 nucleotides in length, but typically they range from 15 to 30 bases. It is the amplification primers that determine target specificity (which segment of DNA will get amplified). The ideal annealing temperature of a particular primer is related to its length and sequence (G+C content). There are two major variables in determining the optimum conditions for a particular PCR reaction 1) the annealing temperature and 2) the Mg++ ion concentration.

2 Both influence the annealing of the primers. If the temperature is too low during the annealing step or if the Mg++ concentration is too high then the primers can bind non-specifically, that is to sequences that are not an exact complementary match to the primer. This results in nonspecific amplification of DNA and the appearance of multiple amplification products upon electrophoresis. In contrast, the temperature during the annealing step can be too high or the Mg++ concentration too low resulting in no amplification of the target DNA at all because the primers fail to bind the target. Each synthesis cycle is composed of three steps (Figure 1): 1) Denaturation. During the denaturation step, the reaction cocktail is exposed to high temperature, usually 95 o C. This high temperature will denature the DNA--meaning the two complementary strands of the DNA molecule unravel, exposing the nucleotide bases. The high temperature of the denaturing step has the added advantage of denaturing proteins and disrupting cells so you don t have to always start with purified DNA as your amplification template, you can often times amplify DNA directly from cell lysates. 2) Primer Annealing. During the second step of each cycle, the temperature is lowered to allow annealing of the primers to their complemantary targets on the DNA template (one for each DNA strand). These are designed to flank the desired target region of your DNA template and serve as the starting points for DNA synthesis by the Taq polymerase. 3) Extension. The reaction cocktail is now brought to the optimum temperature for Taq polymerase (68 to 72 o C). During this step, the Taq will bind to and extend from the priming sites (synthesize a complementary strand of the targeted DNA). Figure 1. Dipiction of the three steps of each PCR cycle denaturation, annealing, and extension (polymerization). Image from Typically, PCR reactions are run for 30 to 35 cycles, which are performed by a specialized machine called a thermocycler that is designed to heat and cool the reaction tubes rapidly. The thermocycler that we will use has a specialized heating block, which can be programmed to generate a temperature gradient across the thermal block during the annealing step. This will allow us to examine the effect of 12 different annealing temperatures upon our PCR reaction and to determine the optimum annealing temperature for your primer pair. In this week s exercise,

3 you will be determining the optimal annealing temperature for and temperature effect on a primer pair targeted to amplify a 1500 bp fragment of the small subunit ribosomal RNA. Variables and Controls Independent Variable: Annealing temperature (35 o C to 60 o C) Dependent Variable: Constant Variables: Resulting PCR product Reaction cocktail, denaturing and extension temperature, cycling step times. Controls Positive: Negative Template DNA that is known to amplify with the primers No template DNA Due to time constraints and limit resources, we will be running positive and negative controls at only one annealing temperature to be assigned in class Prior to this week a clone library of small subunit ribosomal genes was prepared for your class. The library was constructed by performing PCR using the contents of the hindgut from a single termite with primers that anneal to the small subunit ribosomal gene (ssu rdna). Each clone in this library will carry a plasmid that contains a ssu rdna gene from a termite gut organism. Each team will select a single clone from the library from which you will amplify its ssu rdna cloned insert and then prepare a PCR reaction cocktail for sixteen 50ul PCR reactions (12 annealing temperatures, a positive control, a negative control and enough extra to compensate for pipetting errors). The PCR products you generate will then be submitted for DNA sequencing. The sequence from your rdna clone will be used to molecularly identify one termite gut organism and ultimately form the basis of your phylogeny paper. The combined rdna sequences from this year s class with then be used to gain an overall picture of the microbial community in the termite gut (strictly by molecular means, without any culturing of the community). Procedures PCR Materials 1.5 ml Eppendorf centrifuge tubes (Ep tubes) 0.2 ml PCR tubes (12 tubes per strip). Racks for holding the Ep tubes Empty tip boxes for holding PCR tube strips. Clone library (agar plate) Taq polymerase (Taq polymerase) 10X PCR buffer 25 mm MgCl2 Nucleotides (1.25 mm dntps) forward primer (to be assigned in class) reverse primer (to be assigned in class)

4 nuclease-free H 2 O E. coli DNA (template for positive control) Protocol You will be performing a total of 14 PCR reactions. To reduce variation between treatments (different annealing temperatures plus controls), you will make a single reaction cocktail and aliquot it into individual PCR tubes as follows: Single reaction cocktail Cocktail for 16 reactions 30 ul H2O 480 ul 5 ul 10X buffer 80 ul 2 ul 25 mm MgCl2 32 ul 8 ul 1.25 mm dntps 128 ul 2 ul 10 pmol/ul forward primer 32 ul 2 ul 10 pmol/ul reverse primer 32 ul 1 ul Taq 95 (Taq pol enzyme) 16 ul 1 E. coli colony from the termite gut library Use one single colony. 1. Prepare the cocktail and gently vortex. 2. Transfer 49 ml of the cocktail into two PCR tubes to be used as your control tubes. 3. Add 1 ml of nucrease-free H 2 O to the neg. control tube (i.e. no template) and 1 ml of E. coli DNA to the pos. control tube (i.e. known positive template). Record in your notebook the amount (in nanograms) of E. coli DNA you added. 4. You will then add one E. coli colony containing the cloned ribosomal gene to the remaining cocktail. 5. Pick one single colony with a pipette tip and add it to your cocktail. Be sure to pick a visible chunk of the colony, but not any of the agar material. 6. Vortex the cocktail again 7. Aliquot 50 ml of the cocktail into each of 12 PCR tubes. 8. Keep your reactions on ice until each team is ready to place the reactions in the thermocycler. 9. Place the cap strip on the tubes, but do not seal them until they are on the machine. Forcing the lids on with out support may crack the tubes 10. Place the tube strip in the machine. Note that the left side will be the lower annealing temperature. 11. Close the hot top lid and screw the lid slightly tight, but not so tight as to crush the tubes. 12. Choose the program you desire to run. In this case you will be running the program called 16SGRAD 13. Start program by following menu guide. Thermocycler cycling conditions for 16SGRAD 95 o C for 3 min (an intitial high temperature step to make sure the cells are lysed and the DNA denatured) 30 cycles of the following profile:

5 Denaturation: 95 o C for 30 sec Annealing: 40 to 60 o C for 1 min (the exact temp of each tube will be provided in lab) Extension: 68 o C for 3 min Then 68 o C for 5 min to make sure all extensions are completed Then 4 o C -- the machine will hold the tubes at 4 o C until the reaction tubes are removed. The run time for the thermocycler will be about 3 hours so you will not have time to visualize or process your PCR products for sequencing this week. We will remove your reactions from the machine for you, and store them at 20 C. You will run gels on your PCR products the following week (oral presentation week) and then prepare your templates for DNA sequencing the week after that (Week 6). To be done NEXT WEEK. Electrophoresis of your PCR products You will be using agarose gel electrophoresis to visualize your PCR products and observe the effects the different annealing temperatures had on the PCR reaction. Review Agarose gel electrophoresis in the first week s exercise on DNA fingerprinting. 1. Pour a gel with two combs so that you can load all your samples on a single gel. 2. Prepare your samplesfor loading the gel.. Because you will be sequencing your PCR products, you cannot invest your entire reaction cocktail in the gel. Instead, you will use only 10 ul from each of your reactions for the gel as per the steps 3 5 below. 3. Place a piece of parfilm onto the top of an Eptube rack. You will mix your samples on this. 4. Place fourteen 2 ul spots of 10X loading dye on the parafilm. 5. Add 10 ul of your each PCR reaction to a separate spot of loading dye and mix by gentle pipette pumping. 6. Load 5 ul of the 1 kb ladder standard in the left most well of each of the two lanes of wells. 7. Load your 14 PCR reactions. Keep the order from the lowest to the highest annealing temp and make sure you note the order in your lab notebook, including where you load the control reactions. 8. Run the gel, but be sure not to run the top of the gel into the wells of the lower half of the gel. The run time will be limited to about 30 minutes. 9. Visualize and photograph your gel using the UV transilluminator and digital camera.