PCR Enzymes & Related Products

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Upgrade Your PCR Skills

What is PCR?

PCR stands for Polymerase Chain Reaction. It is a widely used laboratory technique to amplify or make many copies of a specific segment of DNA.

How does PCR work?

PCR involves a series of cycles, each involving heating and cooling the DNA sample, enabling the DNA to be repeatedly copied.

Principle of PCR

PCR involves repeated cycles of three main steps:

  1. Denaturation: The double-stranded DNA is heated to a high temperature to separate it into two single strands.
  2. Annealing: The temperature is lowered to allow short DNA primers to bind or “anneal” to their complementary sequences on the single-stranded DNA.
  3. Extension: The temperature is raised slightly to enable the DNA polymerase enzyme to synthesize a new strand of DNA by adding nucleotides to the annealed primers.
PCR Principles

Bioinformatics Tools and Primer Design

Bioinformatics tools play a crucial role in obtaining and analyzing target gene sequence data.

Commonly Used Bioinformatics Tools

  1. NCBI Tools: BLAST, Entrez, PubMed, BLAST2GO
  2. Standalone Tools: EMBOSS, Biopython, ClustalW, MEGA
  3. Web-based Platforms: UCSC Genome Browser, Ensembl, Galaxy

How to Retrieve Gene Sequence from NCBI

  1. Go to NCBI (ncbi.nlm.nih.gov).
  2. Choose the “Nucleotide” database.
  3. Search for gene name or symbol.
  4. Select the correct gene from results.
  5. Find the sequence on the gene page.

Design and Synthesis of Amplification Primers for the Target Gene

Primer design guidelines:

  1. Primer size: 17–25mer
  2. GC content: 40–60%
  3. Align the forward and reverse Tm values (commonly calculated using the nearest neighbor method. Approximate formula: Tm = 2(A+T) + 4(G+C) + 35 – 2N. In the free software Primer3, set 60°C < Tm < 65°C).
  4. Confirm primer specificity (between genes, between species) with a BLAST search.
  5. Consider complementarity of primer sequence (avoid complementarity of 3 bases or more).
  6. Consider the 3’ end sequence (G/C is preferable, avoid T).
  7. Consider the higher-order structure of PCR amplified products.

Types of PCR Enzymes

There are several types of DNA polymerases used in PCR, each with unique properties and applications:

  1. Pol I (Bacterial DNA Polymerase): Pol I is a bacterial DNA polymerase that has a lower fidelity than the α type. However, it has a wider range of suitability for PCR applications. It is commonly used in general PCR reactions.
  2. Alpha Type (α) – DNA Polymerase Derived from Archaea: The α type of DNA polymerase is derived from archaea and has high-fidelity synthesis. It has 3’→5’ exonuclease activity, which allows for proofreading during DNA synthesis. This makes it ideal for PCR applications that require accurate amplification.
  3. Mixed Pol I and α Types: In some PCR applications, a mixture of Pol I and α types is used in an appropriate ratio. This allows for a balance between the broader PCR suitability of Pol I and the high-fidelity synthesis of the α type, making it suitable for a wide range of applications where both accuracy and versatility are needed.

Differences Between Pol I and α PCR Polymerases

Property Pol I (Family A) α (Family B)
Source organism Thermophile bacteria Hyperthermophilic Archaebacterium
3′–5′ exonuclease No Yes
5′–3′ exonuclease Yes No
3′ end of PCR product + dA Blunt
PCR enzyme Taq, EmeraldAmp, etc. PrimeSTAR, Ex Premier, etc.
Example of use Standard PCR High fidelity, Long PCR

Frequently Asked Questions

How do I calculate the melting temperature (Tm) of primers?

The primer melting temperature (Tm) is the estimate of DNA-DNA hybrid stability. Knowing the Tm is critical for determining an appropriate annealing temperature (Ta). A Ta that is too high will result in insufficient primer-template hybridization, leading to low PCR product yield. A Ta that is too low may lead to non-specific product amplification.

Calculation of the Tm of primers shorter than 20 bases can be performed using the Wallace rule:

Tm = 2°C (A+T) + 4°C (G+C)

For accurate estimation of the Tm of primers longer than 20 bases, we recommend using free primer design software such as Primer3.

What primer concentration should be used for PCR?

The final concentration of each primer should be between 0.1 and 0.5 µM. A stock solution of each primer is typically 10–20 µM.

Primer concentrations that are too high increase the chance of mispriming, which may result in nonspecific amplification. Primer concentrations that are limiting can result in extremely inefficient amplification.

How should oligos be purified for PCR?

Standard desalted primers are satisfactory for most PCR applications.

Should I use a three-step or a two-step PCR protocol?

Three-step PCR includes denaturation, annealing, and extension steps. This type of protocol should be used when the Tm of the primers is lower than the extension temperature or is less than 68°C.

If the melting temperature of the primer (Tm) is close to the extension temperature (72°C) or a few degrees lower, consider using a two-step PCR protocol that includes a denaturation step and a combined annealing/extension step. With this protocol, the annealing temperature should not exceed the extension temperature.

Which extension temperature should I use, 68° or 72°C?

A 68°C extension temperature is preferred for two-step PCR and when amplifying longer templates (>4 kb). This lower extension temperature dramatically improves yields of longer amplification products by reducing the depurination rate that influences amplification.

72°C should be used as the extension temperature when performing three-step standard PCR and for amplification of short fragments (<4 kb).

What are the critical factors for amplification of GC-rich templates?

PCR conditions:

  1. Use higher denaturation temperatures (e.g., 98°C as opposed to 94°C or 95°C) to allow complete denaturation of the template.
  2. Keep annealing times for GC-rich templates as short as possible.
  3. Use primers with a higher Tm (>68°C), because annealing can occur at a higher temperature.

The successful amplification of DNA is key to many technologies, whether you’re performing next-generation sequencing, cloning, or genotyping. Your PCR polymerase needs to amplify reliably regardless of your target and sample type. We developed a wide range of the highest-performing PCR enzymes and blends with optimized formulations to meet routine and challenging reaction conditions—from long and accurate PCR to fast PCR to inhibitor-resistant direct PCR.

JadeAMP

DNA Isolation

High-quality starting materials are important for the beginning of all good science. We currently offer DNA extraction kits from our in-house India manufactured brand “DTI” as well as from Macherey-Nagel brand, which is our global partner of Takara Bio for nucleic acid extraction in the bioanalysis range.

Common Nucleic Acid Isolation Methods

Spin column

Spin Column

Magnetic beads

Magnetic Beads

Benefits

  • Ease of use
  • High yield and purity
  • Throughput flexibility
  • No risk of clogging
  • Increased target capture efficiency
  • Rapid collection and concentration of sample

Choose Our DTI Brand for India-Manufactured Range

DTI is the premier in-house brand of DSS Takara Bio India Pvt Ltd., specializing in manufacturing routine laboratory molecular biology products within our state-of-the-art facility, which is ISO 9001 and ISO 13485 approved.

Product offerings

  • Spin Column based Blood DNA
  • Spin Column based Tissue DNA

Choose MN for Your DNA Application

MN has been a reliable partner for DNA purification for years.

Product offerings

  • Tissue DNA
  • FFPE DNA
  • Blood & Plasma DNA
  • Forensic DNA
  • Plant DNA
  • Food DNA
  • Other Organisms & Samples DNA

PCR Enzyme Selection

The successful amplification of DNA is key to many technologies, whether you’re performing next-generation sequencing, cloning, or genotyping. Our large and diverse portfolio of PCR polymerases was designed for success across a variety of applications, from basic and translational research to routine laboratory testing. We create polymerases that simply work faster and better, so you can focus on obtaining the results you need.

PCR Product Offerings

For Routine PCR / Genotyping

  • DTI FabTaq
  • DTI FabTaq HS
  • Premix Taq™ (TaKaRa Taq™ Version 2.0)

For Convenient-Use Dye Plus Master Mix

  • DTI JadeAmp FabTaq Premix
  • DTI CobaltAmp HiFid Taq HS Premix
  • TaKaRa Ex Premier™ DNA Polymerase Dye Plus

For High-Fidelity PCR

  • PrimeSTAR® Max
  • PrimeSTAR® GXL

For High-Yield PCR

  • TaKaRa Ex Taq®
  • TaKaRa Ex Premier™ DNA Polymerase

For Long Fragment PCR

  • TaKaRa LA Taq DNA Polymerase
  • TaKaRa LA Taq® with GC Buffer

For Glycerol-Free Options

  • TaKaRa Taq™ (Glycerol Free)
  • TaKaRa Taq™ HS (Glycerol Free)

For Lyophilised Options

  • Lyophilized HS Taq PCR Master Mix

Accessories

  • dNTP Mixture

Thermal Cycling

DTI FabSpeed Thermal Cycler is designed specifically to enhance PCR efficiency and accuracy. It is equipped with a 7” sensitive touchscreen and a friendly graphic user interface, which makes operation highly intuitive. With flexible ramp rate and gradient temperature control, DTI FabSpeed greatly increases PCR accurate optimization.

Features & Benefits

  • Flexible Ramp Rate Control – from 0.1 to 5.5°C/sec to meet the need for CRISPR-related assays.
  • Fully Adjustable Lid Temperature – can be set between 35 and 120°C for virtually any type of experiment, including NGS pre-treatment.
  • Gradient Optimization – range from 1 to 30°C enables optimal experimental conditions in a single PCR run.

Ordering Information

Brand Catalogue Number Description
DTI Brand TCST-9622 DTI FabSpeed Thermal Cycler

Amplicon Analysis

Agarose gel electrophoresis is a widely used method in PCR workflows for analyzing nucleic acids. It helps separate, identify, quantify, and purify PCR amplicons. Selecting the appropriate products for nucleic acid electrophoresis can greatly enhance and expedite your results.

Our high-quality agarose and markers are designed to support accurate analysis, ensuring you achieve precise and reliable outcomes in your downstream applications.

PCR Product Offerings

DNA Ladder

  • 100 bp DNA Ladder
  • 1 kb DNA Ladder
  • 100 bp DNA Ladder (Dye Plus)
  • 1 kb DNA Ladder (Dye Plus)
  • DL 2,000 DNA Marker
  • DL 5,000 DNA Marker

Agarose Routine

  • Gel and PCR Cleanup

DTI Brand India Manufactured Range

DTI Brand India Manufactured Range

Upgrade Your PCR Skills

Application Standard PCR / General Application Standard PCR / General Application Colony PCR High Yield Genotyping Genotyping
Recommended Product DTI FabTaq DTI FabTaq HS DTI CobaltAmp HiFid Taq HS Premix DTI JadeAmp Max HiFid Taq Premix DTI JadeAmp FabTaq Premix
Choose For Routine Use Routine Use Fast, Routine PCR Streamlined Workflow Value Choice
Properties < 8 kb < 8 kb < 5 kb < 15 kb < 5 kb
5′–3′ Exonuclease Activity YesYesYesYes (Weak)Yes
3′–5′ Exonuclease Activity ––YesYes–
T/A Overhangs or Blunt T/AT/AT/AT/AT/A
Speed 60 sec/kb60 sec/kb10 sec/kb60 sec/kb60 sec/kb
Hotstart –YesYes––
Dye Added ––YesYesYes
Premix ––YesYesYes