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Proofreading

Proofreading

Synthesis of new DNA molecules is carried out by the enzyme DNA polymerase, which adds nucleotides on the daughter strand complementary to the template DNA strand. DNA polymerase has a higher affinity to add the correct base and ensures fidelity during DNA replication. Furthermore, it exhibits proofreading activity during replication, using an exonuclease domain that cuts off incorrect nucleotides from the nascent DNA strand.
Errors During Replication are Corrected by the DNA Polymerase...

Proofreading

Proofreading

Conservative Site-specific Recombination and Phase Variation

Conservative Site-specific Recombination and Phase Variation

Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...

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Engineered Polymerases with Altered Substrate Specificity: Expression and Purification.

Ali Nikoomanzar¹, Matthew R Dunn¹, John C Chaput¹

¹Department of Pharmaceutical Sciences. University of California, Irvine, California.

Current Protocols in Nucleic Acid Chemistry

|June 20, 2017

Summary

This summary is machine-generated.

Researchers developed a protocol to engineer polymerases for synthesizing xeno-nucleic acid polymers (XNAs). This advancement supports synthetic genetics by enabling the study of artificial genetic polymers and their evolution.

Keywords:

aptamer polymerase protein expression and purification xeno-nucleic acid (XNA)

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Area of Science:

Synthetic biology
Biochemistry
Molecular biology

Background:

Xeno-nucleic acids (XNAs) offer diverse chemical functionalities beyond DNA.
XNA synthesis relies on engineered polymerases, often not commercially available.
Recombinant expression in E. coli is a common method for enzyme production.

Purpose of the Study:

To detail a protocol for expressing and purifying engineered polymerases.
To evaluate the activity of polymerases with modified substrate recognition.
To facilitate research in synthetic genetics using artificial genetic polymers.

Main Methods:

Recombinant protein expression of engineered polymerases in E. coli.
Enzyme purification techniques to obtain nuclease-free polymerase.
Activity assays to assess polymerase function and substrate specificity.

Main Results:

A 6-day protocol for polymerase production.
Yields approximately 20 mg of pure, nuclease-free polymerase per liter of culture.
Demonstrated ability to produce functional engineered polymerases.

Conclusions:

The described protocol enables the production of essential engineered polymerases for XNA synthesis.
This method supports the burgeoning field of synthetic genetics.
Facilitates the study of heredity and evolution in artificial genetic systems.