Foodborne disease affects almost 1 in 10 people around the world annually, and continuously presents a serious public health issue (9).
More than 200 diseases have evolved from consuming food contaminated by bacteria, viruses, parasites, and chemical substances, resulting in extensive increases in global disease and mortality rates (9). With this, foodborne pathogens cause a major strain on health-care systems; as these diseases induce a variety of different illnesses characterized by a multitude of symptoms including gastrointestinal, neurological, gynecological, and immunological (9,2).
But why is food contamination increasing?
New challenges, in addition to established food contamination hazards, only serve to compound and increase food contamination risks. Food is vulnerable to contamination at any point between farm and table—during production, processing, delivery, or preparation. Here are a few possible causes of contamination at each point in the chain (2):
Production: Infected animal biproducts, acquired toxins from predation and consumption of other sick animals, or pollutants of water, soil, and/or air.
Processing: Contaminated water for cleaning or ice. Germs on animals or on the production line.
Delivery: Bacterial growth due to uncontrolled temperatures or unclean mode of transport.
Preparation: Raw food contamination, cross-contamination, unclean work environments, or sick people near food.
Further emerging challenges include, more complex food movement, a consequence of changes in production and supply of imported food and international trade. This generates more contamination opportunities and transports infected products to other countries and consumers. Conjointly, changes in consumer preferences, and emerging bacteria, toxins, and antimicrobial resistance evolve, and are constantly changing the game for food contamination (1,9).
Hence, versatile tests that can identify foodborne illnesses in a rapid, versatile, and reliable way, are top priority.
For those of us well versed in traditional, end-point PCR, wrapping our minds and methods around real-time or quantitative (qPCR) can be challenging. Here at Promega Connections, we are beginning a series of blogs designed to explain how qPCR works, things to consider when setting up and performing qPCR experiments, and what to look for in your results.
First, to get our bearings, let’s contrast traditional end-point PCR with qPCR.
Visualizes by agarose gel the amplified product AFTER it is produced (the end-point)
Visualizes amplification as it happens (in real time) via a detection instrument
Does not precisely measure the starting DNA or RNA
Measures how many copies of DNA or RNA you started with (quantitative = qPCR)
Less expensive; no special instruments required
More expensive; requires special instrumentation
Basic molecular biology technique
Requires slightly more technical prowess
Quantitative PCR (qPCR) can be used to answer the same experimental questions as traditional end-point PCR: Detecting polymorphisms in DNA, amplifying low-abundance sequences for cloning or analysis, pathogen detection and others. However, the ability to observe amplification in real time and detect the number of copies in the starting material can quantitate gene expression, measure DNA damage, and quantitate viral load in a sample and other applications.
Anytime that you are preforming a reaction where something is copied over and over in an exponential fashion, contaminants are just as likely to be copied as the desired input. Quantitative PCR is subject to the same contamination concerns as end-point PCR, but those concerns are magnified because the technique is so sensitive. Avoiding contamination is paramount for generating qPCR results that you can trust.
Use aerosol-resistant pipette tips, and have designated pipettors and tips for pre- and post-amplification steps.
Wear gloves and change them frequently.
Have designated areas for pre- and post-amplification work.
Use reaction “master mixes” to minimize variability. A master mix is a ready-to-use mixture of your reaction components (excluding primers and sample) that you create for multiple reactions. Because you are pipetting larger volumes to make the reaction master mix, and all of your reactions are getting their components from the same master mix, you are reducing variability from reaction to reaction.
Dispense your primers into aliquots to minimize freeze-thaw cycles and the opportunity to introduce contaminants into a primer stock.
These are very basic tips that are common to both end-point and qPCR, but if you get these right, you are off to a good start no matter what your experimental goals are.
If you are looking for more information regarding qPCR, watch this supplementary video below.
The first time I performed PCR was in 1992. I was finishing my Bachelors in Genetics and had an independent study project in a population genetics laboratory. My task was to try using a new technique, RAPD PCR, to distinguish clonal populations of the sea anemone, Metridium senile. These creatures can reproduce both sexually and asexually, which can make population genetics studies challenging. My professor was looking for a relatively simple method to identify individuals who were genetically identical (i.e., potential clones).
PCR was still in its infancy. No one in my lab had ever tried it before, and the department had one thermal cycler, which was located in a building across the street. We had a paper describing RAPD PCR for population work, so we ordered primers and Taq DNA polymerase and set about grinding up bits of frozen sea anemone to isolate the DNA. [The grinding process had to be done using a mortar and pestle seated in a bath of liquid nitrogen because the tissue had to remain frozen. If it thawed it became a disgusting mass of goo that was useless—but that is a topic for a different blog.] Since I had never done any of the procedures before, my professor and I assembled the first set of reactions together. When we ran our results on a gel, we had all sorts of bands—just what he was hoping to see. Unfortunately, we realized that we had added 10X more Taq DNA polymerase than we should have used. I repeated the amplification with the correct amount of Taq polymerase, and I saw nothing. Continue reading “Optimizing PCR: One Scientist’s Not So Fond Memories”
Some thermostable DNA polymerases, including Taq, add a single nucleotide base extension to the 3′ end of amplified DNA fragments. These polymerases usually add an adenine, leaving an “A” overhang. There are several approaches to overcome the cloning difficulties presented by the presence of A overhangs on PCR products. One method involves treating the product with Klenow to create a blunt-ended fragment for subcloning. Another choice is to add restriction sites to the ends of your PCR fragments. You can do this by incorporating the desired restriction sites into the PCR primers. After amplification, the PCR product is digested and subcloned into the cloning vector. Take care when using this method, as not all restriction enzymes efficiently cleave at the ends of DNA fragments, and you may not be able to use every restriction enzyme you desire. There is some useful information about cutting with restriction sites close to the end of linear fragments in the Restriction Enzyme Resource Guide. Also, some restriction enzymes require extra bases outside the recognition site, adding further expense to the PCR primers as well as risk of priming to unrelated sequences in the genome.
Scientists have had a love-hate relationship with PCR amplification for decades. Real-time or quantitative PCR (qPCR) can be an amazingly powerful tool, but just like traditional PCR, it can be quite frustrating. There are several parameters that can influence the success of your PCR assay. We’ve highlighted ten things to consider when trying to improve your qPCR results.
A: The simplest way to clone PCR Products is to amplify the product using thermostable polymerases such as Taq, Tfl or Tth polymerase. These polymerases add a single deoxyadenosine to the 3´-end of the amplified products (3´-end overhang), and can be cloned directly into a linearized T-vector.
Q: What if my DNA polymerase has 3´ to 5´ exonuclease activity (i.e., proofreading activity) that removes the 3´-end overhang?
A: To clone PCR products that have been amplified with a polymerase that have proof reading activity into a T-vector, you will need to perform an A-tailing step using Taq DNA polymerase and dATP. Blunt ended restriction digest fragments can also be A-tailed using this method. The method below uses GoTaq Flexi DNA Polymerase (comes with a Mg-free reaction buffer), but any Taq DNA polymerase can be used.
Set up the following reaction in a thin-walled PCR tube:
1–4µl purified blunt-ended DNA fragment (from PCR or restriction enzyme digestion)
2µl of 5X GoTaq Reaction Buffer (Colorless or Green)
2µl of 1mM dATP (0.2mM final concentration)
1µl GoTaq Flexi DNA Polymerase (5u/µl)
0.6µl of 25mM MgCl2 (1.5mM final concentration)
Nuclease-free water to a final volume of 10µl
Incubate at 70°C for 15–30 minutes in a water bath or thermal cycler.
Q: What is a T-vector, and why are they used for cloning PCR products?
A: T vectors are linearized plasmids that have been treated to add T overhangs to match the A overhangs of the PCR product. PCR fragments that contain an A overhang can be directly ligated to these T-tailed plasmid vectors with no need for further enzymatic treatment other than the action of T4 DNA ligase.
The polymerase chain reaction (PCR) has revolutionized modern biology as a quick and easy way to generate amazing amounts of genomic data. However, when PCR doesn’t work, it can be frustrating. At these times, PCR and reverse transcription PCR (RT-PCR) inhibitors seem to be everywhere: They lie dormant in your starting material and can co-purify with the template of interest, and they can be introduced during sample handling or reaction setup. The effects of these inhibitors can range from partial inhibition and underestimation of the target nucleic acid amount to complete amplification failure. What is a scientist to do?
For most molecular biology applications, knowing the amount of nucleic acid present in your purified sample is important. However, one quantitation method might serve better than another, depending on your situation, or you may need to weigh the benefits of a second method to assess the information from the first. Our webinar “To NanoDrop® or Not to NanoDrop®: Choosing the Most Appropriate Method for Nucleic Acid Quantitation” given by Doug Wieczorek, one of our Applications Scientists, discussed three methods for quantitating nucleic acid and outlined their strengths and weaknesses. Continue reading “Methods for Quantitating Your Nucleic Acid Sample”
By clicking “Accept All”, you consent to the use of ALL the cookies. However you may visit Cookie Settings to provide a controlled consent.
If you are located in the EEA, the United Kingdom, or Switzerland, you can change your settings at any time by clicking Manage Cookie Consent in the footer of our website.
Necessary cookies are absolutely essential for the website to function properly. These cookies ensure basic functionalities and security features of the website, anonymously.
This cookie is set by GDPR Cookie Consent plugin. The cookie is used to store the user consent for the cookies in the category "Analytics".
The cookie is set by GDPR cookie consent to record the user consent for the cookies in the category "Functional".
This cookie is set by GDPR Cookie Consent plugin. The cookie is used to store the user consent for the cookies in the category "Other.
The cookie is set by GDPR cookie consent to record the user consent for the cookies in the category "Advertisement".
This cookie is set by GDPR Cookie Consent plugin. The cookies is used to store the user consent for the cookies in the category "Necessary".
This cookie is set by GDPR Cookie Consent plugin. The cookie is used to store the user consent for the cookies in the category "Performance".
6 months 2 days
This cookie is set by the provider Media.net. This cookie is used to check the status whether the user has accepted the cookie consent box. It also helps in not showing the cookie consent box upon re-entry to the website.
This cookie is used to store the language preferences of a user to serve up content in that stored language the next time user visit the website.
Analytical cookies are used to understand how visitors interact with the website. These cookies help provide information on metrics the number of visitors, bounce rate, traffic source, etc.
This cookie is associated with Sitecore content and personalization. This cookie is used to identify the repeat visit from a single user. Sitecore will send a persistent session cookie to the web client.
This domain of this cookie is owned by Vimeo. This cookie is used by vimeo to collect tracking information. It sets a unique ID to embed videos to the website.
1 month 18 hours 24 minutes
This cookie is used to calculate unique devices accessing the website.
This cookie is installed by Google Analytics. The cookie is used to calculate visitor, session, campaign data and keep track of site usage for the site's analytics report. The cookies store information anonymously and assign a randomly generated number to identify unique visitors.
This cookie is installed by Google Analytics. The cookie is used to store information of how visitors use a website and helps in creating an analytics report of how the website is doing. The data collected including the number visitors, the source where they have come from, and the pages visted in an anonymous form.
Advertisement cookies are used to provide visitors with relevant ads and marketing campaigns. These cookies track visitors across websites and collect information to provide customized ads.
1 year 24 days
Used by Google DoubleClick and stores information about how the user uses the website and any other advertisement before visiting the website. This is used to present users with ads that are relevant to them according to the user profile.
This cookie is set by doubleclick.net. The purpose of the cookie is to determine if the user's browser supports cookies.
5 months 27 days
This cookie is set by Youtube. Used to track the information of the embedded YouTube videos on a website.
Performance cookies are used to understand and analyze the key performance indexes of the website which helps in delivering a better user experience for the visitors.
This cookies is set by Youtube and is used to track the views of embedded videos.
This is a pattern type cookie set by Google Analytics, where the pattern element on the name contains the unique identity number of the account or website it relates to. It appears to be a variation of the _gat cookie which is used to limit the amount of data recorded by Google on high traffic volume websites.