Working with specific applications and conditions

All primer pairs used in multiplex PCR should have similar priming efficiencies for their target DNA. This can be achieved by using primers with nearly identical optimum annealing temperatures.

When designing primers, pay special attention the following parameters:

• Homology with the target nucleic acid sequence
• Length
• GC content
• Concentration
• Primer homology (primers should not have homology either internally or with one another, especially at the 3' ends)

Nested PCR is a method that involves re-amplification to improve PCR results. Nested PCR involves designing a new forward-nested (FN) or reverse-nested (RN) primer that is internal to the original primer and can pair with the original partner primer. A very small amount of the primary PCR product is used as a template for PCR with nested primers.

Nested PCR frequently leads to improved yield of the desired PCR product by:

• Eliminating extra bands that may have been present in the initial PCR
• Producing a robust band that may have been weak or invisible in the initial PCR

It is important to note that only a very small amount of the primary product should be used in nested PCR because this template has very low sequence complexity. To start, the primary PCR product can be diluted 1:100, and 1 µl can be used as the template for nested PCR. Also, you may need to reduce the number of cycles to 25–30. The optimal conditions for nested PCR should be determined empirically.

During the PCR denaturation step, all DNA molecules will become single stranded. When the temperature decreases for annealing, three types of duplexes can be formed:

• Homoduplexes—annealing of complementary strands
• Heteroduplexes—cross-hybridization of homologous sequences that may have partial homology
• Duplexes between primers and template

To achieve higher specificity, heteroduplex formation should be minimized by increasing stringency (i.e., increasing the temperature) during the initial PCR cycles. Touchdown PCR increases specificity by using reaction conditions that gradually reduce the annealing temperature. The initial annealing temperature is set to several degrees above the estimated T_m of the primers. In subsequent cycles, the annealing temperature is slowly decreased until it reaches the calculated annealing temperature of the primers (Don 1991). By using a higher annealing temperature in the initial PCR cycles, touchdown PCR favors accumulation of amplicons for sequences with the highest primer-template complementarity, thereby enriching for the most specific amplicons. Transitioning to a lower temperature during subsequent cycles reduces stringency, improving priming conditions with the already enriched, desired template. We recommend performing an initial 5–10 cycles with the higher annealing temperature, and then gradually decreasing the temperature until the optimal annealing temperature, or "touchdown temperature," is reached. For example, if the T_m of your primers is 68°C, the recommended TD-PCR conditions for the annealing temperature are:

• 5 cycles at 72°C, then
• 5 cycles at 70°C, then
• >25 cycles at 68°C

References

Don, R. H., et al. 'Touchdown' PCR to circumvent spurious priming during gene amplification. Nucl Acids Res. 19(14):4008 (1991).

If a PCR product is amplified with a high-fidelity polymerase that generates blunt ends, you can perform A-tailing using Taq polymerase. A brief protocol for adding 3' A-overhangs to PCR products is provided below.

1. Purify the PCR product. Before adding overhangs, it is very important to remove all of the polymerase in the reaction by purifying the PCR product using a PCR purification kit or by phenol extraction and DNA precipitation. This step is critical, since the proofreading activity of any residual DNA polymerase would degrade the A overhangs, thus recreating blunt ends.
2. Prepare the Taq DNA polymerase reaction mix: 

*The A-addition reaction works best when a specific amount of the PCR product is used. The recommended amount is 10–100 ng per 100 bp of the PCR product. This corresponds to 0.15–1.5 pmol of PCR product (see table below).
 

3. Incubate for 20 min at 72°C.

Proceed to TA cloning. For optimal ligation efficiency, it is best to use fresh PCR products, since 3' A-overhangs will gradually be lost during storage.

• Enzymes in the PrimeSTAR series
  These enzymes exhibit substantial 3'→5' exonuclease activity and primarily generate amplification products with blunt ends. Therefore, we recommend using the Mighty Cloning Reagent Set (Blunt End) for blunt-end cloning.
• Taq and Terra enzymes
  Takara Taq, Takara Ex Taq, Takara LA Taq, SpeedSTAR HS, EmeraldAmp, SapphireAmp Fast, and Terra PCR DNA polymerases primarily yield amplification products containing 3'-dA overhangs that can be directly used for TA-cloning. Blunt-end cloning is also possible using the Mighty Cloning Reagent Set (Blunt End).
• All Takara Bio DNA polymerases
  The fragment terminal structure after amplification with a particular polymerase is indicated in the table below. Some polymerases generate a mixture of blunt-end and A-overhang products; other polymerases generate only blunt-end or A-overhang products.

Polymerase	A-overhang	Blunt end
Takara Taq DNA polymerases
Takara Ex Taq DNA polymerases
Takara LA Taq DNA polymerases
Titanium Taq DNA Polymerase
Advantage 2 Polymerase Mix
Advantage GC 2 Polymerase Mix
Advantage HF 2 Polymerase Mix
EmeraldAmp GT PCR Master Mix
EmeraldAmp MAX HS PCR Master Mix
SapphireAmp Fast PCR Master Mix
High Yield PCR EcoDry Premix
High Fidelity PCR EcoDry Premix
Terra PCR Direct Polymerase Mix
SpeedSTAR HS DNA Polymerase
PrimeSTAR HS DNA Polymerase
PrimeSTAR GXL DNA Polymerase
PrimeSTAR Max DNA Polymerase
CloneAmp HiFi PCR Premix
SeqAmp DNA Polymerase
TaKaRa Z-Taq DNA Polymerase
e2TAK DNA Polymerase
PerfectShot Ex Taq DNA Polymerase

The fidelity of a DNA polymerase refers to its ability to accurately replicate a template, or to add the correct nucleotides starting at the 3' end of the primer. The rate of base misincorporation is known as the error rate. PCR polymerases with proofreading activity possess 3'→5' exonuclease activity that can excise incorrectly incorporated nucleotides and replace them with the correct nucleotides.

High-fidelity polymerases are recommended for gene cloning, protein expression, structure-function studies of proteins, cDNA library construction, and next-generation sequencing.

Error rates reported by vendors for polymerases cannot always be directly compared, as different methods are used to measure fidelity. These methods include:

1. Blue-white screening
   This approach is based on phenotypic changes and is widely used since it is fast, relatively simple, and cost effective. The original method for blue-white screening, known as the Kunkel method (Kunkel and Tindall 1987), is based on α-complementation of the lacZα gene that restores β-galactosidase enzyme activity and allows production of a blue color. With this method, colonies derived from lacZα PCR products containing single-nucleotide errors or frameshift mutations typically have a white color, while clones derived from error-free amplicons generate blue colonies.
2. Sequencing approach
   This approach utilizes Sanger sequencing of individual colonies after PCR. The blue-white screening approach can quickly measure polymerase fidelity, however it is not as accurate as the sequencing approach. The blue-white method will not detect silent mutations, single-nucleotide substitutions that do not affect translation. The sequencing method can detect all mutations, and thus is more accurate.

References

Kunkel, T. A. and Tindall, K. R., Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase. Biochemistry 27, 6008–6013 (1987). 

The fidelities of the PrimeSTAR polymerases were measured by Sanger sequencing of individual colonies after PCR, as described below:

• Ten arbitrarily selected GC-rich regions of Thermus thermophilus HB8 genomic DNA were amplified.
• PCR products were cloned into a plasmid vector.
• Multiple clones were selected for each respective amplification product, and the PCR insert was sequenced.

PrimeSTAR Max DNA Polymerase has a fidelity 29X that of Taq polymerase, whereas PrimeSTAR GXL DNA Polymerase has a fidelity 6.5X that of Taq polymerase.

TaKaRa Taq DNA Polymerase and TaKaRa Taq DNA Polymerase Hot Start Version are compatible with inosine-containing primers.

Inosine-containing primers should not be used with PCR enzymes that have 3'→5' exonuclease activity (e.g., PrimeSTAR HS DNA Polymerase, PrimeSTAR Max DNA Polymerase, PrimeSTAR GXL DNA Polymerase, TaKaRa Ex Taq DNA Polymerase, or Takara LA Taq DNA polymerases), nor with Terra PCR Direct polymerase. When using one of the compatible Takara Taq PCR enzymes for degenerate PCR, we recommend using a mixture of degenerate primers with A, T, G, or C at the desired position(s) rather than inosine-containing primers.

PrimeSTAR polymerases provide better fidelity than Pfu DNA polymerase, which is widely considered to be a high-fidelity enzyme. PrimeSTAR Max DNA Polymerase has the highest fidelity; when this enzyme was used to amplify the entire pUC119 plasmid, sequence analysis detected only four mutations out of 370,656 total bases sequenced (an error rate of 0.0010%). In addition, compared to other enzymes, PrimeSTAR Max DNA Polymerase replicates repeat sequences with markedly better fidelity and exhibits a lower rate of template exchange (formation of chimeric molecules) with analogous sequences.

Consider the following enzymes:

1. First choice: PrimeSTAR GXL DNA Polymerase is the most effective enzyme for GC-rich templates, such as bacterial genomic DNA. It facilitates high-fidelity amplifications with very few errors. PrimeSTAR GXL DNA Polymerase has been used successfully to amplify a region with ~70% GC content in a standard reaction using the buffer provided with the enzyme.
2. Second choice: Advantage GC 2 Polymerase Mix is recommended for complex templates containing up to 90% GC content. This polymerase is suitable for fragments up to 6 kb. Advantage GC 2 polymerase used with DMSO and GC-Melt reagent allows amplification of virtually all GC-rich sequences.
3. Third choice: for GC-rich targets that have rigid structures and are difficult to amplify with PrimeSTAR GXL DNA Polymerase, use TaKaRa LA Taq DNA Polymerase with GC Buffer. Try GC Buffer I first; this buffer facilitates the amplification of long products. GC Buffer II is effective for templates with complex higher-order structures, although this buffer is more effective for amplification of shorter products.

PrimeSTAR GXL DNA Polymerase is recommended when both length (>6 kb) and fidelity are factors. Amplification of 30-kb products from human genomic DNA templates has been accomplished with this enzyme. Takara LA Taq DNA polymerase can also be used for long-range PCR; this enzyme is recommended when length and robust amplification are priorities.

We have several PCR enzymes that can be used for fast reactions.

• Speed and fidelity—PrimeSTAR Max DNA Polymerase contains a proprietary elongation factor and exhibits excellent priming efficiency, allowing extension times as short as 5 sec/kb and an annealing time of only 5 sec. This enzyme is recommended for cloning and expression studies.
• High-throughput applications and fast colony PCR—SapphireAmp Fast PCR Master Mix is an economical choice for high-throughput projects. This 2X enzyme premix includes a high-speed polymerase, optimized buffer, dNTP mixture, gel loading dye (blue), and a density reagent. Since it requires an extension time of only 10 sec/kb, colony PCR reactions can be completed in <1 hr for inserts up to 1 kb.
• SNP genotyping and fast long-range PCR—SpeedSTAR HS DNA Polymerase is highly efficient and can reliably perform PCR amplifications with extension times between 10 and 20 sec/kb.

TaKaRa Ex Taq DNA Polymerase and PrimeSTAR GXL DNA Polymerase are effective for amplifying AT-rich target sequences, such as genomic DNA containing introns or AT-rich mitochondrial DNA. We recommend PrimeSTAR GXL DNA Polymerase for amplifying AT-rich templates with high accuracy. In contrast to other PCR enzymes, PrimeSTAR GXL polymerase can amplify targets containing >60% AT content using a standard PCR protocol and the reaction buffer provided.

Note: PrimeSTAR GXL DNA Polymerase cannot be used to amplify bisulfite-treated DNA or other uracil-containing templates. For this application, try TaKaRa EpiTaq HS (for bisulfite-treated DNA).

Several choices are available:

• TaKaRa EpiTaq HS (for bisulfite-treated DNA) is optimized for PCR amplification using bisulfite-treated DNA templates that contain uracil. This enzyme includes a hot-start antibody and is designed for use during methylation analysis, including COBRA and bisulfite-sequencing analyses.
• The EpiScope MSP Kit is designed specifically for methylation-specific PCR (MSP) assays.
• TaKaRa Taq DNA Polymerase and TaKaRa Taq DNA Polymerase Hot Start Version, both of which lack 3'→5' exonuclease activity, may be used in some instances.

TaKaRa Ex Taq DNA Polymerase is exceptionally robust, even in the presence of PCR inhibitors such as polyphenols found in crude DNA extracts from plant tissue. PrimeSTAR GXL DNA Polymerase is recommended when high fidelity is also required. Although PrimeSTAR GXL polymerase generally produces satisfactory results with the standard protocol, doubling the enzyme concentration may improve results if very high concentrations of inhibitors are present.

Alternatively, Terra PCR Direct Polymerase Mix allows direct amplification from crude samples that may contain high levels of PCR inhibitors. Terra PCR Direct polymerase can efficiently amplify a wide range of targets, including GC- and AT-rich targets. This enzyme can also be used for direct PCR from blood samples.

In cases where amplification products cannot be produced with these enzymes, it may be necessary to purify the template DNA.

The Terra PCR Direct FFPE Kit can be used for crude DNA preparation and direct PCR amplification from paraffin-embedded tissue sections.

If it is necessary to extract DNA from paraffin-embedded tissue first, TaKaRa DEXPAT Easy and TaKaRa DEXPAT Reagent enable quick, efficient DNA preparation, even from slides or samples that have been stored for years.

We recommend EmeraldAmp GT PCR Master Mix. This PCR master mix contains a green gel loading dye and density reagent, making it easy to prepare PCR reaction mixtures that can be directly loaded on an agarose gel for electrophoresis after PCR. EmeraldAmp GT PCR Master Mix can amplify targets up to 10 kb in length, including targets that are GC- or AT-rich. Additionally, PCR products generated with EmeraldAmp GT PCR Master Mix can be used directly for restriction enzyme digestion, sequencing, or TA-cloning without purification.

We recommend SapphireAmp Fast PCR Master Mix for colony PCR. This enzyme premix can tolerate a substantial presence of bacterial nucleic acids. Tracking dye and density reagent are included in the master mix, allowing the PCR reaction products to be loaded directly on an agarose gel for electrophoresis.

For the highest sensitivity, we recommend Titanium Taq DNA Polymerase or TaKaRa Ex Taq DNA Polymerase. Both polymerases have been used successfully for highly sensitive genotyping applications.

We recommend Terra PCR Direct Polymerase Mix for amplifying targets from crude extracts or directly from tissues. The recommended amounts of various tissues that should be used for direct PCR are listed below:

• Treated blood: ≤5 µl
• Mouse tail: ≤1 mm
• Mouse ear: ≤1.5 mm²
• Plant leaf: ≤1.2 mm (diameter)
• Paraffin-embedded tissue section: ≤1–1.5 cm²

We recommend TaKaRa Taq DNA Polymerase or Titanium Taq DNA Polymerase for multiplex PCR. View our tech note for more information about using Titanium Taq DNA Polymerase in multiplex PCR for high-throughput genotyping.