What happens when PCR becomes present (it might be even better than meditation.)

PCR exponentially amplifies copies of a DNA sequence. It’s great at producing more DNA for subsequent analysis. For example PCR combined with gel electrophoresis can tell us whether a particular loci is present or absent (Figure 1). It’s also somewhat of a black box – you put your sample in a thermal cycler, program in the cycles’ temperatures, and awhile later come and collect your product. We know what happens in between but we can’t see it happening in real time. Or rather we couldn’t until quantitative PCR, abbreviated as qPCR, came along. qPCR enables us to see the amount of amplified product as thafter every cycle.

What is qPCR?

A qPCR reaction is a PCR reaction with fluorescent dyes added (Figure 2). These fluorescent dyes let off a signal every time a strand of DNA is doubled. Consequently the amount of the fluorescence released during amplification is directly proportional to the amount of amplified DNA. This has some major advantages. By using a fluorescent reporter in the reaction, it is possible to tell if a sequence is present/absent without the second gel step. Also qPCR can accurately tell us the original amount of DNA in a sample. Such information is useful for monitoring the genetic expression of a particular gene and for detecting infectious diseases, cancer, and genetic abnormalities.

What does qPCR look like?

Theoretically all PCR reactions exponentially produce DNA. This means that there is positive linear relationship between the number of cycles and the log of the amount of DNA produced. In reality we can only see a small fragment of this line. In the beginning DNA amplification is too small to accurately observe. In the graph in figure 3 everything below the red line represents signals that are either undetectable or hard to measure because of background noise. At the end of a reaction reagents begin to run out which slows down the reaction. This is seen as a gradual flattening of the line. In between these two areas is the log linear phase. This is the region of the graph where the relationship between the fluorescence and the amount of starting material is the most consistent and measurable. Where the log linear line begins, e.g. where it intersects with the threshold line is known as the Cq (quantification cycle) point.

Figure 2

How does qPCR answer the “how much” question?

The Cq point is defined as the number of cycles required for the fluorescent signal to exceed background levels. It is inversely proportional to the amount of starting target nucleic acid in the sample – the more starting material the lower the Cq value for that sample. A Cq value for a sample can be compared to a standard curve to calculate the exact copy number of the original template. Creating this standard curve entails preparing a dilution series of known concentrations and then plotting the log of the initial template copy number against the Cq generated for each dilution. Comparing Cq values of unknown samples to this standard curve allows the quantification of the initial copy number.

Figure 3

Introduce your students to the power of qPCR with Kit #380 Discovering Quantitative PCR Amplification and Analysis or Kit #381 Break Through! Testing DNA Damage Using Quantitative PCR.

Are you gearing up for end of term exams?

Take a break and read about horizontal gene transfer in the ultimate survivor – the moss piglet. (After all cramming is a little like genetic transformation.)

Figure 1

Recently, tardigrades (known by some as moss piglets and others as water bears) have been making headlines in the field of genetics. What are these excellently nicknamed critters? They are very small aquatic animals. A quick search will show some amazing pictures (and videos). Imagine a less than 1 mm animal with a head attached to a barrel shaped body and four pairs of clawed legs. Interestingly, the hindmost legs are orientated differently from the front six – one useful way to distinguish them from other microscopic animals. Inside they have digestive, excretory, and nervous systems.

These animals are cosmopolitan, which means they can be found everywhere. Tardigrades have been found in hot springs, at the top of glaciated mountains, under layers of ice, and at the bottom of the ocean! As their distribution implies, tardigrades are incredibly resilient. For example, they can handle being cooled to 1 degree above absolute zero (~485°F), heated to 304°F, and dehydrated for 100 years! Scientists even tested them in the vacuum of outer space for 10 days. They survived.

This ability to survive large environmental swings has led scientists to take a close look at tardigrade DNA. More specifically two scientific groups – one from the University of North Carolina and one from the University of Edinburgh – have separately sequenced the genome of the tardigrade Hypsibius dujardini (hip-SIB-ee-us doo-zhar-DEE-nee). The results from these two groups differed significantly, particularly when it comes to the estimated number of horizontally transferred genes.

Horizontal gene transfer is when DNA passes from one organism to another organism through means other than inheritance. The most common forms of horizontal gene transfer include transformation, transduction, and conjugation. UNC scientists identified a huge number of horizontally transferred (HT) genes in their tardigrade sequences – around 6,600 genes or 17.5% of the genome. However, scientists at Edinburgh called this large number into question. They estimated that the number of HT genes in their sequenced data was less than 500 and suggested that the UNC group’s numbers were skewed by contamination.

Contamination is a challenge when sequencing organisms like tardigrades. The problem is not just proper lab sterilization. The DNA of symbiotic and pathogenic microorganisms that live in places like an organism’s gut inevitably get sequenced along with the host’s DNA despite many precautionary steps. In order to distinguish between “foreign” genes that come from these organisms and “foreign” genes that are a result of horizontal transfer scientist’s at UNC re-sequenced the areas around potential HT genes. This re-sequencing showed that the foreign genes were on the same DNA strand as established tardigrade genes and thus were likely part of the tardigrade genome. However, this additional analysis was done for only a small subset (107) of the identified HT genes.

Resolving whether the number of horizontally transferred genes in the tardigrade genome is in the tens, hundreds or thousands will take time. Luckily, both labs have made their data and methodology available to the larger research community. This will allow experts from around the world to participate in the discussion.

While this debate continues we encourage you to check out these amazing creatures! The best way to do this is to combine a small amount of moss (collected from a backyard or park) and a small amount of distilled water in a petri dish or similarly sized bowl. Allow the moss to soak overnight and then squeeze the water from the moss. Take this water and put it back in the petri dish or in a microscope slide. You should be able to see tardigrades in this water sample, especially under a dissecting microscope between 5 and 30 power magnifications. If you are interested in carrying out some horizontal gene transfer on your own we recommend checking out our exciting transformation kits (#221, #223/AP08, #225, #300 and #301).

Figure 1

Edvotek

Is your school about to go on spring break? Are you worried about your electrophoresis gels diffusing? Last summer, we tested stain longevity and now we have it down to a science! Here are our best tips.

At Edvotek, we field tech questions every day. Our dedicated scientists have heard it all and one of our most common topics of inquiry is staining agarose gels post electrophoresis and more specifically, how long the gel will keep following staining.

All of our stained gels will maintain a good band to background contrast for 5+ days using a simple three step method:

  1. Following electrophoresis and staining, place each gel in a small ziplock storage bag.
  2. Add an additional 1-2 ml of electrophoresis buffer.
  3. Place in the fridge.

The key to this procedure is to only put a tiny amount of buffer into the bag – 1-2 ml allows the gel to stay hydrated but does not allow the DNA to diffuse out. We’ve had great success using this method with FlashBlue™, InstaStain® Ethidium Bromide, and SYBR®Safe stains.

A final option is to use a highly diluted FlashBlue stain (1:150) to soak the gel for 3 or more hours. This method is outlined in many of our protocols, but may require an additional destaining step of 2-3 hours when stained for more than 12 hours.

This blog is moving! Come visit us at our new home.

Hi all —

We know it’s been a while since we’ve updated this page, but it’s been for a good reason — the blog is now on our recently redesigned website! Please stop by and check it out at www.edvotek.com/News. We’ll be updating it regularly with the same quality content that we have been posting here at edvotek.info.

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Looking forward to seeing you in our new location!

Best,

The Edvotek Blog Staff