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?
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?
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
How does qPCR answer the “how much” question?
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
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.
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.)
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.
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.
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
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.
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).
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:
Following electrophoresis and staining, place each gel in a small ziplock storage bag.
Add an additional 1-2 ml of electrophoresis buffer.
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.
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.
Looking forward to seeing you in our new location!