DNA Amplification, PCR & qPCR
Product Overview
In order to study or detect individual genes or specific DNA regions or mutations of interest, it is often necessary to obtain a large quantity of nucleic acid for study. Rather than isolate a single copy of the target DNA from a large number of cells, it is often more useful to generate multiple copies of a target from a single molecule of DNA or mRNA, via an in vitro amplification method.
As the Polymerase Chain Reaction (PCR) is the most common DNA amplification method in molecular biology, NEB’s product portfolio features a large selection of polymerases geared towards this powerful method. As the first company to sell Taq DNA Polymerase to the research market, the first to discover a PCR-stable, high-fidelity DNA polymerase, and the first to provide reagents for PCR performed in space, NEB has a long history of developing reliable and convenient PCR tools. This commitment has continued with the recent development of OneTaq® DNA Polymerase for robust routine PCR and Q5® High-Fidelity DNA Polymerase for robust, ultra high-fidelity PCR (>280 X Taq fidelity). Both product lines have been developed to tolerate a variety of complex templates without experiencing a loss of performance on high-AT or high-GC targets. A variety of NEB polymerases, including OneTaq, Taq and Q5, also benefit from novel aptamer-based hot start technology that does not require a separate activation step.
For experiments where detection and quantification is required instead of isolation, quantitative PCR (qPCR) uses real-time fluorescence to meaure the amount of a DNA target present at each cycle during a PCR. The most common methods of generating a fluorescent signal are by use of hydrolysis probes (e.g., TaqMan®), or a double-stranded DNA binding dye, (e.g., SYBR® Green). Ideally, qPCR master mixes should be evaluated for high reaction efficiency over a wide linear dynamic range, and low variation between repeated reactions across a broad variety of sample types. NEB’s Luna® product line has been developed to simplify your qPCR reagent selection while accomplishing best-in-class performance.
Despite the ubiquitous nature of PCR and qPCR, it may not be the best option for all amplification needs. For point of care and other diagnostic applications, sequence-specific isothermal amplification methods, that eliminate the need for thermocycling, have been particularly useful. Instead of heat, these methods typically employ a strand-displacing DNA polymerase, like Bst DNA Polymerase, Large Fragment, to separate duplex DNA. To address some of the limitations of current isothermal amplification techniques, NEB has developed the next generation Bst, Bst 2.0 and a WarmStart® version of this enhanced polymerase, which enables room temperature reaction setup, yet is fully active at temperatures greater than 50°C.
RNA molecules can also be detected and manipulated through amplification via the use of reverse transcriptases (RT), which are RNA-dependent DNA Polymerases. RTs polymerize a strand of DNA that is complimentary to the original RNA template and is referred to as cDNA. This cDNA can then be further amplified through PCR, qPCR or isothermal methods as outlined above or detected in a single reaction using one-step RT-qPCR or RT-LAMP.
Nucleic acid amplification is a foundational process in molecular biology and, as a testament to its utility, new protocols and modifications are being developed constantly. At NEB, our goal is to use our understanding of enzymology, and dedication to providing high-quality products, to offer reagents for all of your applications. Please visit the application pages below to learn more.
DNA Amplification, PCR & qPCR includes these areas of focus:
Isothermal Amplification
- Loop-Mediated Isothermal Amplification
- Whole Genome Amplification & Multiple Displacement Amplification
- Strand Displacement Amplification & Nicking Enzyme Amplification Reaction
- Helicase-dependent Amplification
- Recombinase Polymerase Amplification and SIBA
- Nucleic Acid Sequenced Based Amplification and Transcription Mediated Amplification
PCR
- Routine PCR
- High-Fidelity PCR
PCR & Reaction Cleanup
- PCR & Reaction Cleanup
Polymerases for DNA Manipulation
- Polymerases for DNA Manipulation
qPCR & RT-qPCR
- Probe-based qPCR & RT-qPCR
- Two-Step RT-qPCR
- Dye-based qPCR & RT-qPCR
RT-PCR & cDNA Synthesis
- cDNA Synthesis
- RT-PCR
Site Directed Mutagenesis
- Site Directed Mutagenesis
Specialty PCR
- Hot Start PCR
- Long Range PCR
- Fast PCR
- Extraction-Free PCR
- Multiplex PCR
- Bisulfite Sequencing
- Polymerases for NGS Library Preparation
Whole Genome Amplification
- Whole Genome Amplification
FAQs for DNA Amplification, PCR & qPCR
- What is touchdown PCR?
- Which NEB DNA polymerases can incorporate fluorescently-labeled nucleotides during PCR?
- Polymerases and Amplification FAQs
Protocols for DNA Amplification, PCR & qPCR
- PCR Using NEBNext® High-Fidelity 2X PCR Master Mix (NEB #M0541)
- PCR Using Q5® Hot Start High-Fidelity DNA Polymerase (NEB #M0493)
- PCR Using Q5® Hot Start High-Fidelity 2X Master Mix (NEB #M0494)
- PCR Using Q5® High-Fidelity 2X Master Mix (NEB #M0492)
- PCR Optimization (E0555)
- Protocol for a Routine PCR (E0555)
- Protocol for a PCR reaction using NEBNext® Q5® Hot Start HiFi PCR Master Mix (M0543)
- Luna® Universal qPCR Master Mix Protocol (NEB #M3003)
- Luna® Universal One-Step RT-qPCR Kit Protocol (E3005)
- Luna® Universal Probe One-Step RT-qPCR Kit Protocol (E3006)
- Luna® Universal Probe qPCR Master Mix Protocol (M3004)
- Luna® Probe One-Step RT-qPCR Kit (No ROX) Protocol (NEB# E3007)
Application Notes for DNA Amplification, PCR & qPCR
- Optimized Integration of New England Biolabs® Loop-mediated Isothermal Amplification (LAMP) Reagents with Axxin ISO Instruments
- Primer Monitor: an online tool to track SARS-CoV-2 variants that may impact primers used in diagnostic assays
- The Luna 4X RT-qPCR Mix and Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit enable high throughput, automated detection workflows
- Multiplex PCR using Q5® High-Fidelity DNA Polymerase
- Key considerations for optimal lyophilized reagent development
- Detection of the Omicron variant mutation at position 26,270 in the SARS-CoV-2 E gene using the SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit
- Optimized conditions for the CDC Influenza SARS-CoV-2 (Flu SC2) Multiplex Assay using Luna One-Step RT-qPCR Reagents
- Accelerating DNA Construction to Protein Expression A Rapid 1-Day Workflow Using NEBridge Golden Gate Assembly
- Optimizing rapid isothermal workflow for SARS-CoV-2 with WarmStart® LAMP with UDG
- Facilitating Purification and Detection of Viral Nucleic Acids from Milk
- Enabling Whole Genome Amplification from picogram quantities of genomic DNA with the phi29-XT WGA Kit
- Universal Annealing Temperature in PCR and its Impact on Amplification Results
- A Practical Guide to Analyzing Nucleic Acid Concentration and Purity with Microvolume Spectrophotometers
- Multiplex real-time PCR detection of monkeypox virus using Luna® qPCR Reagents
Tools & Resources
Brochures
- PCR Reagents Brochure
Web Tools
- Polbase
Selection Tools
- DNA Polymerase Selection Chart
- Reverse Transcriptase Selection Chart
Troubleshooting Guides
- PCR Troubleshooting Guide
- Luna® qPCR Troubleshooting Guide
- Luna® One-Step RT-qPCR Troubleshooting Guide
Usage Guidelines
- Guidance on Choosing Sample Input Amounts when Using the Monarch Total RNA Miniprep Kit
- Guidelines for PCR Optimization with OneTaq® and OneTaq® Hot Start DNA Polymerases
- Guidelines for PCR Optimization with Taq DNA Polymerase
- Guidelines for PCR Optimization with Thermophilic DNA Polymerases
- Optimization Tips for Luna® One-Step RT-qPCR
- Optimization Tips for Luna® qPCR
- General Guidelines for Successful RNA Purification Using the Monarch Total RNA Miniprep Kit
Posters
- Molecular Diagnostics for Gastrointestinal Parasites and Impact on Intestinal Microbiota in Rural Agentinian Children (2015)
- A Single-tube, Low Input Protocol for Long Read Sequencing (2019)
- Utilizing Lyophilized LAMP Reagents in Rapid Molecular Assays
Publications related to DNA Amplification, PCR & qPCR
Legal Information
Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. All other trademarks are the property of their respective owners. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
