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Monday, June 4, 2012

Hot Start Taq DNA Polymerase, Types, Advantages, Commercially available Hot Start Taq


Taq DNA Polymerase is a thermostable enzyme(Molecular Weight 94 KDa) which is widely used in Polymerase chain reaction (PCR), for amplifying short stretches of DNA. Kary mullis invented Polymerase chain reaction in 1983.

Taq DNA Polymerase was first isolated from thermophilic bacteria Thermus aquaticus, inhabiting hot springs. The most significant feature of Taq polymerase is that the enzyme is active at higher temperature.

Types of PCR enzymes
  1. Taq Polymerase: Isolated from single genus of bacteria Thermus aquaticus, thermophilus, filiformis, brockianus.
  2. Pfu Polymerase:* Isolated from small group of Archea bacteriaPyrococcus furiosus, woseii, Thermococcus litoralis.
Gene encoding taq polymerase is cloned and expressed in E.coli.

The advantage of Pfu polymerase is that it has 3' – 5' exonuclease proof reading activity, it corrects nucleotide incorporation errors, which means the amplified product will have lesser error than the product amplified
used normal Taq Polymerase.it produces blunt end PCR product. But it is expensive and slower requires more cycling time.

Before going into the details of Hot Start Taq and Hot Start PCR, let's look into the basics of Polymerase Chain Reaction and the problems associated with Taq Polymerase / non Hot Start Taq Polymerase

Polymerase Chain Reaction has 3 steps:
  1. Initial Denaturation
  2. Annealing
  3. Extension
Polymerase chain reaction (PCR) works by denaturing the double stranded DNA at higher temperature around 94oC. This step is called initial denaturation step. Due to higher temperature hydrogen bond breaks and resulting in double stranded DNA to separate out into two single strands.

Once the double stranded DNA separates out, the primes attaches to the single stranded DNA, annealing takes place at a temperature 5oC below the primer Tm (melting temperature).

Primer binding or annealing takes place during the annealing step and next step is the extension of primers by Taq polymerase at 72oC. Because both strands are copied during PCR, there is an exponential increase of the number of copies of the gene. Suppose there is only one copy of the wanted gene before the cycling starts, after one cycle, there will be 2 copies, after two cycles, there will be 4 copies, three cycles will result in 8 copies and so on. (2n ; n denotes the cycle number).

Problems associated with normal PCR / non Hot Start Taq Polymerase 

1. Specificity
2. Selectivity
3. Yield

Taq polymerase shows activity at room temperature, once the PCR mix is prepared primers can bind each other forming primer-dimer this can get amplified by the Taq polymerase which reduces the target DNA amplification.

Non-specific binding of primers to the DNA also can result in the amplification resulting in non specific PCR product formation.

Hot Start PCR:

It is a PCR technique which helps to reduce the non-specific amplification in the initial setup stages, in this technique modified Taq polymerase called Hot Start Taq Polymerase is used.

Hot Start Taq Polymerase




HotStar Taq DNA Polymerasse - Qiagen Image Source

Hot Start Taq Polymerase is a modified version of Taq DNA polymerase, Hot start Taq polymerase is formulated with heat labile monoclonal antibodies or chemical that, at room temperature, effectively neutralize DNA polymerase activity. Full enzyme activity is regained upon denaturation of the antibody or the chemical during the initial denaturation step. Using Hot start Taq DNA polymerase, hot start step can be easily incorporated into PCR protocols which are already optimized with Taq DNA polymerase, with little or no modification of cycling parameters or reaction conditions.

Types of Hot Start Taq Polymerase

1. Antibody Based Hot Start Taq
2. Chemically Modified Hot Start Taq
3. Wax Bead based Hot Start Taq
4. Sequester Primers

*Antibody Based Hot Start Taq Polymerase*

Hot Start Taq polymerase is formulated with a monoclonal antibody is bound to the active site of the enzyme taq polymerase there by preventing the catalysis through protein-protein interaction. By raising the temperature (~95oC) antibody denatures leaving the active site free.

Low activation energy is required to remove the antibody.

95oC for around 1-5 mins is enough to remove the antibody attached to the
Taq Polymerase.

*Chemically Modified Hot Start Taq Polymerase*

A small organic compound which inhibits the activity of Taq polymerase, at higher temperature the chemical compound strips off and making the taq polymerase active. Chemically modified Taq must be activated by breaking inter molecular bonds and this requires high activation energy, 10 – 15 mins at 95oC is needed to remove the chemically bound compound.

*Wax Bead Based Hot Start Taq Polymerase*

Taq Bead Hot Start Taq Polymerase is a product from promega. *Taq*Bead™ Hot Start Polymerase consists of spherical beads of wax containing Taq DNA polymerase. Taq polymerase will be available for action only when thetemperature reaches around 60oC (at this temperature wax bead melts) there
by preventing non specific amplification and primer-dimer formation.

*Sequester Primers*

It is a novel method which uses a single-stranded DNA binding protein to sequester primers at lower temperatures, making them unavailable for extension by the polymerase. Following the initial denaturation step, the binding protein is inactivated and the primers are free to participate in the amplification reaction. This method targets the primers and not the polymerase, it is portable to a variety of thermostable polymerases. The "primer-sequestration" technique effectively blocks non-specific product formation before thermal-cycling and enhances end-point and real-time PCR reactions.

Advantages of Hot Start Taq polymerase

The advantage of using Hot Start Taq polymerase is that:

- Higher yields with high specificity and sensitivity.

- Reduced / No primer dimer formation: Hot Start Taq polymerase helps to reduce the formation of primer-dimer (primers hybridize together and amplifies), which competes with the amplification of the target DNA there by reducing the target DNA amplification.

- Set up reactions at room temperature.

- Can let reactions sit at room temperature for at least 24 hours before the cycling protocol is started.

- No long initial denaturation step in cycling protocol (For antibody based Hot start Taq)

Disadvantages of Hot Start Taq polymerase:

There are few disadvantages for Hot Start Taq Polymerase

1.The antibody is from hybridoma cells which can contaminate reactions with mammalian DNA.

2.The removal of chemical blocking group on the polymerase typically requires initial denaturation times of greater than 10 minutes which causes heat-damage to DNA samples.

Commercially available Hot Start Taq Polymerase

HotStarTaq Plus DNA Polymerase, Chemically Modified, Qiagen

GoTaq® Hot Start Polymerase, Antibody Based, Promega

TaqBead™ Hot Start Polymerase, Wax Based, Promega

Platinum®Taq DNA Polymerase, Antibody Based, Life Technologies

EpiMark® Hot Start *Taq* DNA Polymerase, NEB

JumpStart™ Taq DNA Polymerase, Sigma-Aldrich

TaKaRa Ex Taq® DNA Polymerase Hot-Start, Antibody Based, Takara

HotStart-IT Taq DNA Polymerase, Sequester primers, USB

FastStart Taq DNA Polymerase, Roche Applied Science

illustra Hot Start Maser Mix, Sequester primers, GE

Ampli Taq Gold, Chemically Modified, Applied Biosystems

Cheetah HotStart Taq DNA Polymerase, Chemically Modified, Biotium Inc.

KAPATaq HotStart DNA Polymerase, Antibody Based, Kapa Biosystems

References:

Abgene Technical Resources

Promega Technical References

Qiagen Technical Resource

NEB Technical Refernces

Genesis Biotech Inc Resources

Sigma-Aldrich

Kapa Biosystems

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