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Sunday, October 20, 2019

Lac Operon : Gene Regulation in Ecoli (Prokaryotes)

Operons are groups of genes clustered in the bacterial DNA regulating the expression of functionally related proteins.

 In E. coli half of the genes are clustered into operons which are involved in particular metabolic pathways.

Lac operon model was proposed by Jacob & Monad.

Lac Operon is responsible for the transport and metabolism of lactose.

Tuesday, October 15, 2019

Taq DNA polymerase and its key features

Taq DNA polymerase is one the important component in the PCR Mastermix. Role of Taq DNA polymerase is to amplify the DNA strands. Let’s look at the features of Taq DNA polymerase in detail:



  1. Specificity
  2. Fidelity
  3. Processivity / Speed
  4. Thermostability

Specificity

One of the major challenge in PCR is the specific amplification of target DNA. Nonspecific amplification occurs due to the extension of misprimed targets and primer dimer formation. This can impact overall sensitivity and specificity of PCR assay.

To avoid amplification of misprimed template or primer dimer one option is to setup the pcr reaction in cold condition or add Taq polymerase as a last component in the reaction mixture. This method will help in reducing extension of primer dimer, misprimed template to some extent.

As the Taq polymerase has activity even at low temperature (activity will be less compared to its optimal temperature) extension of mispriming can occur during the reaction setup. To prevent this Hot-Start Taq polymerase has been developed.

Hot Start Taq polymerase will be active only after an activation step, which is much higher than the reaction setup temperature, thereby preventing extension of primer dimer and misprimed template.

Fidelity

Fidelity is the proof reading activity of DNA polymerase. This feature is essential for correct replication of DNA (without error). Cloning and site directed mutagenesis requires replication of DNA without errors. High fidelity polymerases are engineered for this applications.

DNA polymerase has two activities:

  • 3’ – 5’ proof reading activity
  • 5’-3’ Polymerase activity
3’-5’ exonuclease activity is the one determines the fidelity. This is also known as the proof reading activity. The ability to correct misincorporated nucleotides.

Due to polymerase activity, Taq DNA polymerase is able to incorporate nucleotides to the primed template. During polymerization, misincorporated nucleotides will be corrected by proof reading activity of the taq polymerase.

Taq DNA polymerase’s fidelity is often expressed as the inverse of error rate. No of misincorporated nucleotides per total number of nucleotides polymerized.

Processivity

Processivity of the taq polymerase is its speed or nucleotide incorporation rate per single binding event. High processivity Taq polymerases are required for amplifying longer targets in short time. It is beneficial in amplifying template DNA having higher GC content and also in amplifying crude samples, samples which are minimally processed.

New engineered versions of Taq polymerase are highly processive compared to the regular taq DNA polymerases.

Thermostability

PCR cycling involves repeated heating and cooling of the reaction mixture. Enzyme used in the reaction should withstand high temperature. Taq DNA polymerase isolated from bacteria living in hotsprings has revolutionized the PCR process.

Thermostability is the ability of taq DNA polymerase to withstand higher temperature. Taq DNA polymerase isolated from thermophilic bacteria is known for its thermostability. Half-life of the Taq polymerase reduces above 90°C.

The above features of Taq DNA polymerase makes it a versatile enzyme to be used in the polymerase chain reaction.