DNA Polymerases fused to nonspecific DNA binding domains e.g. Phusion (TM)

In the Public Domain since

16 Aug 2020

Description

DNA polymerases are the copy machines of biology and are very essential to modern biological engineering. Thermostable DNA polymerases are the core enzyme component of polymerase chain reaction (PCR), the process by which specific sequences of DNA can be rapidly copied from a template and amplified exponentially to generate the genetic material required for DNA assembly, expression, mutation and transformation reactions. For most PCR applications, it is desirable for the DNA to be copied with high speed and high fidelity. The speed and fidelity of DNA polymerization depend in large part on the properties of the DNA polymerase used in the reaction. Polymerization speed, for example, depends both on the intrinsic maximum rate at which the polymerase can catalyze the incorporation of nucleotides into the new DNA strand and on the number of nucleotides the polymerase incorporates, on average, before stochastically dissociating from the DNA (the polymerase’s processivity).

In 2000, Yan Wang patented a method for increasing the processivity of DNA polymerases (as well as other enzymes that ‘modify nucleic acids’ such as RNA polymerases, topoisomerases, gyrases, and methylases). By attaching a DNA polymerase to a protein domain that binds to double-stranded DNA (dsDNA) of any sequence, the rate at which the polymerase falls off of the DNA strand it is replicating could be greatly reduced. This increase in processivity translated to DNA replication that was faster and of higher fidelity. The Phusion polymerase, which is a genetic fusion of the Pyrococcus furiosus thermostable DNA polymerase and the Sso7 DNA binding domain, is the most famous product covered by this patent.

To conclude, some very good DNA polymerases are coming off patent in the US on August 16, 2020. Get excited! If you want to start making or distributing these polymerases, Barrick lab has sequences and protocols for expressing and purifying your own Pfu-Sso7D fusion enzymes. Pipette Jockey has a blog post showing the performance of this enzyme in different buffer conditions and on different DNA templates.

Why it is important

DNA polymerases have various rates of processivity, i.e. the rate at which they can synthesize new strands of DNA from an existing DNA template. Polymerase Chain Reaction (PCR) programs must allow enough time for DNA polymerases to complete synthesis of new DNA strands for each cycle. This can take a long time if the desired DNA contains thousands of nucleotides. However, with an addition of nonspecific DNA binding domains to your DNA polymerase of choice, you can easily reduce the waiting time for synthesis by 50-75% and get your DNA products much quicker.

Sso7D domain peptide sequence:

ATVKFKYKGE EKEVDISKIK KVWRVGKMIS FTYDEGGGKT GRGAVSEKDA PKELLQMLEK QKK

Sso7D domain nucleotide sequence:

GCAACCGTAA AGTTCAAGTA CAAAGGCGAA GAAAAAGAGG TAGACATCTC CAAGATCAAG AAAGTATGGC GTGTGGGCAA GATGATCTCC TTCACCTACG ACGAGGGCGG TGGCAAGACC GGCCGTGGTG CGGTAAGCGA AAAGGACGCG CCGAAGGAGC TGCTGCAGAT GCTGGAGAAG CAGAAAAAG

Abstract

This invention provides for an improved generation of novel nucleic acid modifying enzymes. The improvement is the fusion of a sequence-non-specific nucleic-acid-binding domain to the enzyme in a manner that enhances the ability of the enzyme to bind and catalytically modify the nucleic acid.

Category

Enzymes > DNA Polymerases, Enzymes

Related Inventors

unknown

Source

US Patent No 6627424

Patent Title: Nucleic Acid Modifying Enzymes

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