Polymerase Chain Reaction (PCR) is an essential tool for biological analysis. This molecular biology technique gained newfound recognition when it was thrust into mainstream attention during the onset of the COVID-19 global pandemic; and PCR testing became the standard tool for detection of the disease-causing virus (SARS-CoV-2).
Since its invention in the 1980s, PCR has become instrumental for many applications such as medical testing, biological research, quality control, forensic analysis, and environmental monitoring.
PCR technology utilizes the biological DNA replication machinery to amplify small quantities of genetic material to facilitate downstream analysis—such as microbiological detection and genetic fingerprinting. Therefore, it is important to carefully process and prepare PCR samples to minimize contamination and increase available material.
Equally important is understanding all the key steps, interactions and chemical reactions that take place during the amplification of genetic material to generate usable and quantifiable results. In this presentation, we will look at the basics of PCR and the practical aspects of sample preparation, chemical reagent selection, and types of processing that can improve or amplify the PCR workflow.
Despite its mistaken reputation as a difficult technique to master, PCR is, in fact, a simple tool that offers accurate and rapid results. Whether you are involved in clinical diagnostics, medical research, bioprocessing, vaccine and therapeutics development, food and beverage, agriculture, or botanicals testing, you can use PCR to deliver quick and consistent results.
Antylia Scientific™ (formerly known as Cole-Parmer) and Spex® have a range of innovative tools that can be leveraged for rapid, reliable, and reproducible PCR analysis.
Speakers
Patricia Atkins — Senior Applications Scientist at SPEX® CertiPrep
Patricia graduated from Rutgers University in NJ and started her career as a laboratory supervisor for Ciba Specialty Chemicals. In 2008, Patricia joined SPEX CertiPrep within their Certified Reference Material’s division. As a senior application scientist, she spends much of her time researching industry trends and developing new reference materials. She is an experienced speaker and has presented at numerous conferences, including NACRW, NEMC, Pittcon and AOAC. She is also a published author and her work has appeared in various journals and trade publications, including Spectroscopy, LCGC and Cannabis Science and Technology, where she is a columnist for analytical issues in botanicals testing.
Alan Katz, PhD — Director, Global Product Manager - Chemicals
Alan holds a BA in Chemistry from the University at Buffalo and a PhD from Princeton University. His specialties include organic, computational, heterocyclic, and synthetic chemistry, drug discovery, biological assay design, structure-based molecular modeling, and lab automation. He has enjoyed combining his expertise with his interest in computer programming to develop a system to assist in drug discovery. At SPEX CertiPrep, he oversees the manufacturing and quality control of certified chemical reference materials, including inorganic and organic chemical standards, NPD and R&D.
Eric Smith – Applications Scientist at Spex SamplePrep
Eric holds a BS in chemistry from Kean University. His current role at Spex SamplePrep involves managing all phases of application development including specifications, data collection, literature research, reporting and presentations. He serves as applications scientist and an internal ISO 9001 auditor; a technical expert, traveling domestically and internationally for customer visits and tradeshows. Prior to joining Spex, Eric worked as a R&D and QA chemist and currently also serves as an instructor at the International Center for Diffraction Data and the London X-ray Consulting Group.
Lea Anderson-Smith, PhD – Director, Engineering (R&D) at Spex SamplePrep
Lea Anderson-Smith holds a BS in Chemistry from the University of Wisconsin and a PhD in Inorganic Chemistry from MIT. She is currently Director of Engineering for Spex SamplePrep and Katanax equipment and accessories, managing design and development of new products and updates to existing products. She also managed operations for the Spex SamplePrep business for many years and maintains an active role in technical support for customers and service of customer-owned equipment. She works closely with the product management and commercial teams in identifying new product and application opportunities with the goal of helping scientists in their work.
Madalina Enache – Product Sales Manager (EMEA) – PCRmax®, Techne®, and Argos Technologies®
Madalina holds a BSc (with Hons) in Biology and a MSc in Microbiology. She joined Antylia Scientific™ three years ago, when it was known as Cole-Parmer, as part of the application sales team. She has risen through several commercial roles, focused on PCR products, to become the EMEA Product Sales Manager for our PCRmax, Techne, and Argos Technologies brands.
Questions & Answers from the live event
What types of samples require cryogenic grinding instead of room temperature homogenization?
Cryogenic grinding is used for tough samples that cannot be ground atroom temperature, such as bone, teeth, and hair. This technique is frequently used for extracting DNA from forensic or archeological samples and has also been used with marine samples such as whale blubber and a shark tooth.
What is the typical homogenization time on the Spex® Geno/Grinder®?
Typically, 1 to 3 minutes depending on the sample type andquantity. With the Spex DNA Isolation Kit, samples were homogenized from 1to 3 minutes. For homogenization longer than 1 minute, the process was runin cycles: 1-minute run time with a 20-second rest period between cycles.
Can Spex provide guidance on selecting the correct disruption tubes, grinding media, and homogenization protocols?
Yes. A document with guidelines on selecting the right products for yoursamples is available for download from this webinar. Our technical expertsare also happy to help you in determining homogenizing conditions and choosing the best homogenizer for your needs.
Are there other dyes that can be used other than the ones mentioned in the presentation?
The PCRmax® ECO 48 system is factory calibrated for use with SYBR®,FAM™, HEX®, VIC®, ROX®, Cy®5 and can also be used with any qPCR fluorophores overlapping with these calibrated dyes.
Can you over cycle a sample? If so, what happens?
Yes. Over cycling (or aggressive bead beating) can result in shearing the DNA/RNA creating fragments which can decrease the yield.
Can one use normal autoclaved pipette tips instead of RNAse free ones? How big is the mistake?
We would suggestnot to do that because it is not possible to guarantee the removal of all the condensation from the tips. Also, if you are pipetting µL volumes, you will decrease the chances of achieving accuracy.
When choosing a region of the target to amplify, which one will work better: shortor long PCR products? Any suggestions?
It is better to choose very short amplicons (60 to 90 bp) because you wouldwant to complete the run as fast as possible.
Do you offer primers as well? Can youcustomize primers?
We do not offer primers. Currently we offer Delta Seek detection kits. Each kit is specifically designed by our bioinformatics team to ensure the broadest possible detection profile and detection of all clinically relevant strains and subtypes. All test kits are validatedin-house on multiple qPCR platforms to ensure cross-platform functionality.
Which kind of precautions should be considered in using the Geno/Grinder DNA Extraction machine to avoid cross-contamination?
In reference to the 2010 Spex Geno/Grinder, using individual tubes such as 2 mLper sample will avoid cross-contamination of samples along with good laboratory practices.
Do you supply a kit for pathogen BCC USP <60>? Have you ever able to determine the absence of BCC pathogen in a product using your qPCR?
We have the PCRmax DNA Burkholderia cepacia complex (all species), with mastermix for the BBC.
Many modern qPCR protocols use only two-step protocol (denature, anneal/extend) vs. traditional three-step protocol (denature, anneal, extension). Will preamplification enhance performance (amp efficiency, nonspecific amp) in this type of protocol?
The two-step protocol reduces the occurrence of primer-dimers in theno-template control but also permits an improved control of the temperature. If you have limited samples, then reamplification is a powerful technique. The efficiency depends on the reagents. It is important to use a reagent design forpreamplification.
Have you had experience with the Geno/Grinder workingwith algae samples, specifically cyanobacteria (blue-green algae)?
Lysing of bacterial cells can be done in standard 96-well titer plates with 400to 600 µl silica grinding beads or in 2 mL tubes. See the application note below:
Lysing of Bacterial Cells in the Geno/Grinder
With kind permission of Russell H. Vreeland, West Chester University, January 2003
The Geno/Grinder was tested to determine if this technology could be used to lyse bacterial cells. Standard 96-well titer plates were used with 400 to 600 μl silica grinding beads (Molecular Biology Grade, cat. no. 2166).The delivery of the beads into each cell of the titer plate can be accomplished in a number of ways. In this case micropipette tips were filled to the mark with grinding beads, and each tip emptied into a titer plate well. This technique will deliver approximately 0.4 grams of silica beads per well. Other delivery systems are commercially available. Experiments were performed with 250 to 500 ml of bacteria: in one case the gram-negative, salt tolerant Halomonas elongata, and in the other, gram-positive Bacillus. Both organisms were grown to late log state at 37°C. with shaking, then harvested via centrifugation at 7000 RPM for 10 minutes. 0.4 grams of silica beads were added to each well, and the plate was sealed. The filled titer plates were then shaken in the Geno/Grinder at a setting of 1450 strokes per minute, in one-minute intervals for periods ranging from one minute to twelve minutes.Culture filtrate from the shaken plates was then measured for optical density at 260 nm. The average data for three separate “well sets’ is shown in Figure1.
Conclusion
The Geno/Grinder is capable of lysing bacterial cells in 96-well titer plates with 400 to 600 μl silica beads as grinding media. With increased grinding time culture filtrate shows a definite increase in optical density, indicating release of nucleic acids during grinding.Grinding times of 6 to 9 minutes appear to be suitable for producing measurable amounts of nucleic acid.
How do you scale up PCR amplification from 100 uL to 500 uL?
Starting point is to optimize the PCR reaction and find the bestconditions. Also, you will have to know if your PCR machine can work with these volumes. Many PCR systems are calibrated for a fixed volume and particular thickness of plate.
For an unknown sample taken from a crime scene for example, do you have to find the target primer first? How do you do that?
In this case STR (Short Tandem Repeat) loci are amplified using sequence specific primers. These primers are design to amplify STR loci from any individual.
In the grinding media, are there nucleic acid stabilizers?
The grinding media are inert materials with no stabilizers. The stabilizing of nucleic acids should be done with appropriate buffers for the intended extraction. Spex chemists can help you select buffers and grinding media appropriate for your analysis.
Generally, what is the recommended maximum number of cycles to amplify a product of our interest?
Number of cycles is sample dependent. Typical protocol uses 1 to 3 cycles with 1 to 3 run intervals. For conventional PCR, only perform the number of cycles required to amplify the product. For qPCR,there may be requirements in IFUs to perform a specific number of cycles to generate amplification curves and calculate Cq values. In general, no more than 40 cycles should be required to identify a positive amplification. In both cases, running more cycles than necessary increases the risk of amplifying non-specific products.
Can we use Geno/Grinder for cells such as mammalian cells or yeast?
Yes. Lysing of mammalian cells or yeast can be done in standard 96-well titer plates with one 4 mm grinding ball per well, or in a 2 mL tube with 400 µm silica or zirconia beads. Sensitive tissue samples can be lysed in Spex SamplePrep Cryo-Blocks(2 mL or 5 mL format) to prevent sample degradation for RNA extraction.
The Geno/Grinder was tested to determine if this technology could be used to lyse bacterial cells. Standard 96-well titer plates were used with 400 to 600 μl silica grinding beads (Molecular Biology Grade, cat. no. 2166).The delivery of the beads into each cell of the titer plate can be accomplished in a number of ways. In this case micropipette tips were filled to the mark with grinding beads, and each tip emptied into a titer plate well. This technique will deliver approximately 0.4 grams of silica beads per well. Other delivery systems are commercially available. Experiments were performed with 250 to 500 ml of bacteria: in one case the gram-negative, salt tolerant Halomonas elongata, and in the other, gram-positive Bacillus. Both organisms were grown to late log state at 37°C. with shaking, then harvested via centrifugation at 7000 RPM for 10 minutes. 0.4 grams of silica beads were added to each well, and the plate was sealed. The filled titer plates were then shaken in the Geno/Grinder at a setting of 1450 strokes per minute, in one-minute intervals for periods ranging from one minute to twelve minutes.Culture filtrate from the shaken plates was then measured for optical density at 260 nm. The average data for three separate “well sets’ is shown in Figure1.