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In this molecular biology laboratory, students learn the steps of PCR with an emphasis on primer composition and annealing temperature, which they manipulate to test the effect on successful DNA amplification. Students design experiments to test their hypotheses, promoting a discovery-based approach to laboratory teaching and development of critical-thinking and reasoning skills.
The commercial availability of kits has made laboratories utilizing PCR more common in high school and undergraduate science classes. Parameters for these experiments are often standard and preset. Students run the reactions without having a true appreciation for the critical experimental details required to amplify a specific segment of DNA.
The PCR cycle involves three steps: denaturation, primer annealing, and primer extension. Each of these steps requires incubation of the reaction mixture at different temperatures. This breaks the hydrogen bonds between the nucleotide base pairs bp and separates the two strands of DNA. The early innovators of PCR needed to optimize this procedure. Initially, fresh DNA polymerase had to be added after each denaturation step.
Eventually, a thermally stable form was discovered in the hot springs bacteria Thermus aquaticus Taq , hence the term Taq DNA polymerase. Each incubation period required the transfer of test tubes by hand from one temperature to another until the advent of the thermal cycler, which regulates cycling temperatures automatically. Researchers supply their own primers, which are designed to anneal to a specific DNA sequence, and the DNA template to be amplified.
An ideal PCR will be specific, generating one and only one amplification product, be efficient, yielding the theoretical two fold increase of product for each PCR cycle, and have fidelity, reproducing the exact sequence of the template.
Each of these parameters is affected by variables within the PCR reaction mixture such as buffer components, cycling number, temperature, and duration of each cycling step, primer composition, and DNA template. In this laboratory exercise, students use two sets of primers to determine optimal annealing temperature on PCR product formation to optimize for efficiency of amplification.
We use this exercise in a cell physiology laboratory course for upper-division undergraduates. It is also appropriate for AP Biology courses, where funding for more advanced laboratory exercises may be available. This set of experiments focuses on the amplification of two PCR products: one for claudin-2 and one for claudin The claudins are components of tight junctions found between intestinal cells and are involved in creating a permeability barrier so that substances cannot pass from the lumen of the intestine to the blood.
In these experiments, students. An ideal PCR possesses high specificity one and only one product , efficiency good exponential yield , and fidelity an accurate product.
Adjusting these variables will maximize one parameter over another, and thus there is a compromise depending on your purpose. Fidelity is of primary importance when the purpose is to sequence a particular DNA. In quantitative PCR, used for evaluation of gene expression, specificity and efficiency are also important.
Primer length and sequence are critical in amplifying PCR products with specificity and efficiency Dieffenbach et al. The stability of the primer-DNA template duplex is measured by its melting temperature T m , the temperature at which half of the primer-DNA duplex dissociates to become single-stranded DNA. Primer length is typically between 18 and 22 nucleotides.
Table 1 shows the primers used in this exercise to amplify intestinal cDNA for claudin-2 and claudin Students can calculate the optimal annealing temperature on the basis of primer compositions and design an experiment to test different temperature ranges in order to determine the optimal annealing temperature.
The experimental protocol to test actual annealing temperature is described below, and variations are suggested so that instructors can guide students to create their own hypotheses and adapt the experiment to test other variables that students can manipulate. Students can be grouped to test different hypotheses, or a consensus can be reached whereby one hypothesis will be tested by all groups.
The laboratory is divided into three modules. In the third module, the PCR products are analyzed by separation through agarose gels. The class is divided into groups of two or three students, depending on class size. The entire laboratory exercise takes 3—4 weeks, assuming a 3-hour laboratory per week, but each module can be carried out separately so that time can elapse between modules.
A schematic diagram of the exercise is represented in Figure 1. Schematic diagram of experiments used in each module.
A Module 1: preparation of cDNA using reverse transcriptase. C Module 3: agarose gel electrophoresis of PCR products.
Alternatively, mouse intestinal RNA can be purchased from suppliers Amsbio, catalog no. M; or Zyagen, catalog no. A kit from Invitrogen catalog no. MD; or mouse colon cDNA, catalog no. MD and use at a concentration of 10 ng per PCR mixture. Primer sequences for claudin-2 and claudin are shown in Table 1, with details concerning composition and annealing temperatures.
On the basis of class discussions, they can set up the range of annealing temperatures to be tested to determine actual annealing temperature of each set of primers. The database can be used to give students a list of primers to calculate annealing temperatures for, and can also be used to choose different sets of primers to be tested by different groups of students. RR into a 0. In our exercise, we tested 12 different annealing temperatures, so we prepared a 12X reaction mix in a 1.
Students can adjust the number of annealing temperatures chosen, depending on the primers used. If a gradient cycler is not available, the reaction can be run several times in a regular PCR cycler by changing the annealing temperature for each run. After studying the basics of DNA synthesis and PCR, there are many variations on the exercise that instructors might challenge students to examine.
In addition to annealing temperature, variables such as length of primer, concentration of primers and cDNA, and cycle number can also be tested.
After the agarose gel has solidified, it is placed in the buffer chamber filled with 1X TAE buffer so that the gel is submerged, and the comb is gently removed. The cover is placed on the chamber, the electrodes appropriately connected positive—red, negative—black , and the gel is run at 90 V for 90 minutes or until the blue dye is three-quarters of the way toward the end of the gel.
We stain the gel using InstaStain Ethidium Bromide paper Edvotek because this reduces potential exposure to ethidium bromide and is safer for student use. The agarose gel is layered over an ethidium bromide sheet, a second sheet is place over the gel, and a light weight is placed over the gel.
After 10—15 minutes, the sheets are removed and the gel is visualized under ultraviolet light. Separating the PCR products through an agarose gel and staining with ethidium bromide Figure 2 shows one clear band at the expected length for each primer set: bp for claudin-2 and bp for claudin As the temperature deviated from the observed optimal annealing temperature, either decreasing or increasing, the amount of product decreased proportionally.
The claudin primers were able to produce the expected PCR product over a wider range of annealing temperatures than the claudin-2 primers because the claudin primers have a higher T m , which allowed for a more stable primer-DNA duplex than the claudin-2 primers, thus supporting primer elongation at higher temperatures. To assess student learning, the first laboratory begins with a pretest consisting of 20 multiple-choice questions designed to test the student's knowledge of DNA and PCR.
The details of each laboratory module are described, and variables that affect PCR are listed by the class. This allows students to understand the importance of optimization in experimental protocols. Students often perform laboratory exercises without giving thought to the painstaking work involved in development of the protocol, and without fully comprehending and analyzing the outcomes of their experiments Phillips et al.
The instructor and students should explore what variables each group will test and the basis of their hypothesis. A posttest consisting of the same questions is given at the beginning of module 1 to assess both understanding of the concepts and preparation for the lab exercise.
The questions include numerical calculations for CG ratio and annealing temperature. In addition, at the end of module 3, each lab group is required to submit a lab report written in scientific format that includes calculated data on the primers used and images of the agarose gels. This set of laboratory exercises introduces students to DNA amplification using PCR in a way that demonstrates the underlying principles of PCR with emphasis on parameters that influence it.
Toggle navigation. Frequently asked questions Our Scientific Applications Support team has assembled a list of frequently asked questions to help you find answers quickly. How do you calculate the annealing temperature for PCR? The annealing temperature T a chosen for PCR relies directly on length and composition of the primers. One consequence of having T a too low is that one or both primers will anneal to sequences other than the intended target, because internal single-base mismatches or partial annealing may be tolerated.
This can lead to nonspecific PCR amplification and will consequently reduce the yield of the desired product. Conversely, when T a is too high reaction efficiency may be reduced, because the likelihood of primer annealing is reduced significantly. Optimal annealing temperatures give the highest product yield of the correct amplicon. Nucleic Acids Res 18 21 —
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