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Comparing Hot Start vs. Standard PCR
by K Chico, Jessica Piczon
Preliminary hot start PCR approaches were achieved by withholding a key component during reaction setup, as in polymerase, dNTPs, primers, or Mg2+, and then introducing them once the reaction mixture reached a desired, elevated, temperature. These methods use physical, solid, barriers like wax pellets to segregate reaction components until the hot start activation temperature is achieved. At this step, the wax melts and the amplification reaction proceeds as normal, providing for a substantially easier and safer, uninterrupted, PCR process.
Over time, these barriers have been simplified through the creation of pre-molded wax beads that encapsulated reagents which are then released at a certain temperature. More recent developments include the use of microfluidic devices to precisely control or inhibit the addition of components into a PCR reaction.
Another hot start approach is to alter polymerase to effectively block molecular activity until hot start activation. This has been performed through the use of antibodies, aptamers, chemical modifications, protein fusions, temperature-sensitive amino acid point mutants, and temperature dependent ligands which initially bind to polymerase prior to amplification. In this method, the inhibitory molecule is selectively denatured once a certain reaction temperature is reached. After, the fully active polymerase can effectively initiate the polymerization reaction.
The most recent development in hot start PCR approaches incorporates the use of thermolabile chemical modifications to oligonucleotide primers and dNTPs. One notable modified primer construct includes those that have the ability to improve hybridization selectivity. Other primer modifications include those that are removable by 3'-5' exonuclease activity, UV irradiation, or even thermal deprotection. Similarly, dNTPs have been modified with thermolabile groups, which are subsequently removed once a certain reaction temperature is reached.
Hot Start PCR
Chapter 22 - Subtractive and nonsubtractive methods for the analysis of gene expression
Original created on January 9, 2024, last updated on January 9, 2024
Tagged under: PCR, DNA, polymerase, amplification
Polymerase chain reaction (PCR) is a versatile nucleic acid amplification process used across many fields, including molecular biology and forensics. Traditional PCR setups employ a mixture of two specific oligonucleotide primers, a thermostable polymerase, dNTPs, a magnesium-containing reaction buffer, and template sources from either DNA or RNA.
One common issue related to unreliable PCR performance is the incidence of off-target amplifications that result from nonspecific primer binding and extension. These issues are thought to occur during the cooler, less stringent, thermal cycling conditions at which many polymerases are still partially active.
A number of techniques, collectively termed “hot start” PCR approaches, have been devised to reduce these off-target amplifications; the goal is to block polymerase extension at lower temperatures, until the primer annealing temperature is reached. For some applications, hot start PCR may be the only way to successfully amplify the desired products while maintaining a high yield and excellent specificity.
Table 1. Summary of the components required for PCR
| PCR Component ▲ ▼ | Function ▲ ▼ |
| DNA Template | This is the sample DNA that contains the target sequence to be amplified. |
| Thermostable DNA polymerase | The enzyme that catalyzes the formation of new DNA strands complementary to the target sequence. Commonly used polymerases include TaqDNA polymerase and PfuDNA polymerase. |
| Primers (forward and reverse) | Short, single-stranded DNA sequences that hybridize to the sample DNA and start the process of replication. Primers are designed to complement the sequences at the beginning and end of the DNA template intended for amplifying. |
| Deoxynucleotide triphosphates (dNTPs) | These are the "building blocks" from which the DNA fragments will be synthesized and are commonly available in a ready-to-use format, such as ReadiUse™ dNTP Mix *10 mM* (Cat No. 17200) |
| Reaction buffer containing magnesium | This provides a stable environment for the PCR reaction. It has a suitable pH of 8.0 to 9.5 and is fortified with magnesium chloride, a co-factor of DNA polymerase. |
Simplified visualization of primer annealing and chain extension during PCR. Starting at a primer that is attached to the complementary location on the template DNA, polymerase will assemble a new strand from dNTPs present in the reaction mixture. This is identical in both standard as well as hot-start PCR, but the process prior to this step varies. Various methods exist to prevent the activation of polymerase below a desired temperature, in order to minimize unintended amplified products. Illustration made in BioRender.
A number of techniques, collectively termed “hot start” PCR approaches, have been devised to reduce these off-target amplifications; the goal is to block polymerase extension at lower temperatures, until the primer annealing temperature is reached. For some applications, hot start PCR may be the only way to successfully amplify the desired products while maintaining a high yield and excellent specificity.
Variations of Hot-Start Approaches
Preliminary hot start PCR approaches were achieved by withholding a key component during reaction setup, as in polymerase, dNTPs, primers, or Mg2+, and then introducing them once the reaction mixture reached a desired, elevated, temperature. These methods use physical, solid, barriers like wax pellets to segregate reaction components until the hot start activation temperature is achieved. At this step, the wax melts and the amplification reaction proceeds as normal, providing for a substantially easier and safer, uninterrupted, PCR process.
Over time, these barriers have been simplified through the creation of pre-molded wax beads that encapsulated reagents which are then released at a certain temperature. More recent developments include the use of microfluidic devices to precisely control or inhibit the addition of components into a PCR reaction.
Another hot start approach is to alter polymerase to effectively block molecular activity until hot start activation. This has been performed through the use of antibodies, aptamers, chemical modifications, protein fusions, temperature-sensitive amino acid point mutants, and temperature dependent ligands which initially bind to polymerase prior to amplification. In this method, the inhibitory molecule is selectively denatured once a certain reaction temperature is reached. After, the fully active polymerase can effectively initiate the polymerization reaction.
The most recent development in hot start PCR approaches incorporates the use of thermolabile chemical modifications to oligonucleotide primers and dNTPs. One notable modified primer construct includes those that have the ability to improve hybridization selectivity. Other primer modifications include those that are removable by 3'-5' exonuclease activity, UV irradiation, or even thermal deprotection. Similarly, dNTPs have been modified with thermolabile groups, which are subsequently removed once a certain reaction temperature is reached.
Products
Table 2. Available DNA quantifying reagents for PCR
| Product Name ▲ ▼ | Ex (nm) ▲ ▼ | Em (nm) ▲ ▼ | Unit Size ▲ ▼ | Cat No. ▲ ▼ |
| Cyber Green™ [Equivalent to SYBR® Green] *20X Aqueous PCR Solution* | 498 | 522 | 5x1 mL tests | 17591 |
| Cyber Green™ [Equivalent to SYBR® Green] *10,000X Aqueous PCR Solution* | 498 | 522 | 1 mL | 17592 |
| Cyber Green™ Nucleic Acid Gel Stain [Equivalent to SYBR® Green] *10,000X DMSO Solution* | 498 | 522 | 100 µL | 17604 |
| Cyber Green™ Nucleic Acid Gel Stain [Equivalent to SYBR® Green] *10,000X DMSO Solution* | 498 | 522 | 1 mL | 17590 |
| Cyber Orange™ Nucleic Acid Gel Stain [Equivalent to SYBR® Green] *10,000X DMSO Solution* | 496 | 539 | 1 mL | 17595 |
| Gelite™ Green Nucleic Acid Gel Staining Kit | 254 or 300 | Long path green filter | 1 kit | 17589 |
| Gelite™ Orange Nucleic Acid Gel Staining Kit | 254 or 300 | Long path green filter | 1 kit | 17594 |
| Helixyte™ Green dsDNA Quantifying Reagent *200X DMSO Solution* | 490 | 525 | 1 mL | 17597 |
| Helixyte™ Green dsDNA Quantifying Reagent *200X DMSO Solution* | 490 | 525 | 10 mL | 17598 |
| Helixyte™ Green Fluorimetric dsDNA Quantitation Kit *Optimized for Broad Dynamic Range* | 490 | 530 | 200 tests | 17645 |
Table 3. Deoxynucleotides (dNTPs) for use in PCR, real-time PCR, and reverse transcription PCR
References
Hot Start PCR
Chapter 22 - Subtractive and nonsubtractive methods for the analysis of gene expression
Original created on January 9, 2024, last updated on January 9, 2024
Tagged under: PCR, DNA, polymerase, amplification