Three steps of PCR

How do all these components come together to amplify a specific piece of DNA? There are 3 basic steps to PCR: Denaturation, Annealing, and Extension. Let’s explore each of these steps in much greater detail. It should be noted, all of this is taking place in a piece of equipment called a thermocycler that can be programmed to change temperatures after different periods of time. Our actual reactions of PCR are placed into small plastic PCR tubes. These tubes hold the components of our PCR and allow for the temperature to change, letting the reaction proceed. When setting up our PCRs, we typically do it on ice (as we’ll see in lab, we have ice buckets that are easy to insert tubes into) and then move to the thermocycler. 

The first step in PCR is Denaturation. Our template DNA is double stranded, which impedes the primers from binding to their complementary regions in our DNA of interest. Therefore, the first step is to separate the double stranded structure into two distinct single strands. Denaturation is typically done by increasing the temperature to roughly 94 degrees Celsius (note this may change slightly depending on the protocol) which breaks the hydrogen bonds between the nitrogenous bases in the two strands of DNA. 

The second step in PCR is Annealing. Now that our DNA is single stranded, our primers should be able to bind to the appropriate regions of the DNA. For primers to be able to bind though, the temperature needs to be much lower than it was in our Denaturation step: typically, around 55 degrees Celsius. This temperature will vary slightly depending on the nucleotide composition of our primer set (typically annealing works better after optimizing the binding temperature of our primers, we have computer software that can closely predict this, yet we conduct several PCRs at various annealing temperatures to help determine which annealing temperature is best for our set of primers). At the end of the annealing step, we have primers bound to the template DNA on each strand. Note that one primer is complementary to the top strand of DNA and one primer is complementary to the bottom strand of DNA. The concentration of our primers is much higher than the concentration of our template strands. Thus is it most likely that a primer will anneal to a template strand rather than the two template strands annealing back together. 

The third step in PCR is Extension. Our thermostable DNA polymerases become the star of the show in this very important step. Using the template DNA and dNTPs, DNA polymerase creates another strand of DNA that is complementary to the template strand. DNA polymerases adds nucleotides to the 3' end of the primer, putting in the nucleotide that is complementary to the template strand at this position. This will happen on both strands of our original template DNA (figure 2). Traditionally, the DNA polymerases are optimally active at 72 degrees Celsius. Thus, this step typically happens at a temperature of 72 degrees Celsius. 

These three steps of PCR result in an additional copy of DNA being produced (for each molecule of template DNA); however, we don’t stop with just one cycle of these 3 steps. Typical PCR reactions go for 30 or more cycles, creating massive amounts of DNA in a relatively short amount of time. As depicted in figure 2, as each cycle continues, we enrich our PCR products with the region of interest (in other words, it’s not just the original strand that is being used as a template, but also the PCR products from the last cycle or even an earlier cycle). 

Once the PCR is complete, we place our tubes back on ice and can do a variety of things including freezing our sample for later analysis, sending off a small amount for sequencing the DNA, inserting it into a plasmid, or we can analyze our sample immediately with the help of our next topic: Gel Electrophoresis.Experimental Design with PCR

When thinking of PCR and experiments we’ll perform, we need to think scientifically. One aspect to PCR is ensuring that we have designed appropriate controls. Controls are essential parts of an experiment that we may use to compare PCRs to ensure they were performed correctly (or can help in terms of comparison, depending on the template samples of PCR). In general, controls may have no treatment, a standard treatment, or be a type of placebo, it’s used to compare various treatments and/or conditions in our experiment. 

For PCR, it is good to have positive and negative controls. For the negative control, we typically use the same PCR components without the template DNA. This is a great way for ensuring the components we are using aren’t contaminated with DNA from another source that could potentially invalidate our results. For a positive control, ideally, it is best to include a template DNA that you know will be amplified by the chosen primers. Sometimes, this is not possible. For example, at our archealogical dig site, we don't know what DNA we have. In this case, we could amplify a specific region of a gene that is conserved among many organisms, like Beta Actin or Glyceraldehyde Dehydrogenase (glycolytic enzyme).

Conclusion

PCR has revolutionized the molecular biology field and has allowed us to amplify a DNA in a wide variety of applications from crime scene investigation to characterizing evolutionary relationships in phylogeny. We once again think about our archaeological dig and the old sample we found. We managed to amplify several different regions of the DNA and we can send these PCR products to be sequenced. Once we know the nucleotide sequence, we can use computer programs to compare these sequences with other known sequences from organisms. We’ve found that the more similar two sequences are, the more closely related they are evolutionarily speaking. For example, the DNA from a dog and a wolf would be more similar than the DNA from a dog and a human. This is just one example of an important use of PCR. One thing is certain: whatever field of biology you participate in after our workshop, PCR will be a crucial part of it. 

Supplemental Information:

What is DNA? 

As we ease into the revolutionary method of PCR, we must first recognize the most important component of the PCR: DNA. Deoxyribonucleic Acid (DNA) is an important part of our cells. DNA is the genetic material, storing the instructions for life (typically in the form of genes that contain the information needed to make specific proteins in our cells). DNA is stored in the nucleus (along with some found in the mitochondria and the chloroplast depending on cell type/organismal origin) and has a special double stranded, double helix structure. All of the DNA in an organism is called its genome and can serve important functions clinically, diagnostically, and even forensically. See figure 1 for more details.

Looking for some videos on PCR? Here's a few!

1. PCR Introduction

2. PCR Basics

3. PCR Principles

V. Overall dCAPS procedure to genotype for the ag-1 mutation

A. DNA isolation (Appendix I)

1. Isolate DNA from individual plants; make sure and include wild type and ag-1 homozygous controls. 

2. PCR (Appendix II)

a. PCR the controls (wild type DNA, ag-1 DNA) and the unknown DNAs using primers ag-1F and ag-1R. PCR should generate a 142bp DNA fragment. Confirm this by running 5ml of the PCR product on a NuSieve 3:1 agarose gel. (Figure 4)

b. ag-1F: 5'-GATATATTAACATATGTTGATAAATCACTTA-3'

c. ag-1R: 5'-ATAGAATTACCTTCTTGGATCGG-3'

Genotyping of the AG locus in the model plant Arabidopsis

I. Genotyping

A. Genotyping is a process that identifies genetic differences between individuals by examining the DNA sequences of these individuals.

1. Here we will genotype using a technique called dCAPS (derived cleaved amplified polymorphic sequences), that uses PCR, restriction enzyme digestion, and agarose gel electrophoresis.

2. Genotyping allows us to identify individuals that are heterozygous for a particular mutation. Because most mutations are recessive, such individuals usually have a wild type phenotype.

II. Model organisms 

A. Model organisms are species studied extensively by scientists to understand basic biological processes.

1. They have certain advantages that make them good for experimental studies. Scientific progress can be fast with such organisms because many scientists work with them.

2. Organisms like the bacterium Escherichia coli (E.coli), the yeast Saccharomyces cerevisiae, the fruitfly Drosophila melanogaster, the zebrafish (Danio rerio), and the plant Arabidopsis thaliana have helped us uncover the molecular basis of various biological processes. 

3. Because many biological processes have a common evolutionary origin, insights from such studies should be applicable to other organisms.

4. Much of our understanding of plant biology has come through the use of a model plant called Arabidopsis thaliana (usually just referred to as Arabidopsis) which is a member of the mustard family.

III. Arabidopsis

A. Arabidopsis is a small weed related to important vegetable crops like cabbage, broccoli, and collards.

1. It grows in many different regions and climates, including Europe, Asia, Australia, and North America.

2. It has several advantages compared to other plants.

a. Because it is a small plant that only gets to be 30-40 cm high, we can grow lots of these plants in a small space.

b. Arabidopsis has a short generation time of about 6 weeks from seed to seed. This makes doing genetics on this plant feasible.

c. A single Arabidopsis plants produces a lot of seed (~10,000/plant).

d. It can be grown in soil, or on defined nutrient agar plates, or floating in liquid media. 

e. Arabidopsis is self-fertilizing but we can do crosses for genetic characterizations.

f. Arabidopsis can be transformed easily.

g. Arabidopsis has a sequenced genome, which consists of 135 million base pairs composed of about 27,000 genes.

IV. AGAMOUS (AG) gene

A. AG is a gene required for the specification of stamen and carpel identity in Arabidopsis flowers. 

1. A wild-type Arabidopsis flower consists of four concentric rings (whorls) of organs with sepals in whorl one, petals in whorl two, stamens in whorl three, and carpels in whorl four. ag mutants produce sepals in whorl one, petals in whorls two and three, and the fourth whorl is converted into a new flower (which repeat the pattern sepals, petals, petals). ag mutant flowers lack stamens and carpels and thus are both male and female sterile and do not set seeds (Figure 3).

2. This mutant has to be maintained in the heterozygous state (ag-1/+ ); such plants have a wild type appearance and thus can't be visually distinguished from wild type siblings. To identify plants that are heterozygous for the ag-1 mutation (i.e. ag-1/+ or ag-1/AG), we can use a technique called dCAPS to genotype the AG locus to determine if the plants are heterozygous for the ag mutation (ag-1/+).