Eureka AIR delivers breakthrough ideas for toughest innovation challenges, trusted by R&D personnel around the world.

Polymerase

a polymerase and polymerase technology, applied in the field of dna polymerases, can solve the problems of limiting the use of unnatural or modified nucleotide bases and the applications they enable, the use of polymerase enzymes for direct incorporation of dye labelled nucleotides, and the inability to complete the substitution of every reactive nucleotide, etc., to achieve the effect of reducing discrimination and high detection agent labeling

Inactive Publication Date: 2013-11-07
MEDICAL RESEARCH COUNCIL
View PDF1 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention is a modified version of a method called compartmentalised self-replication (CSR) that reduces the discrimination against dye-labeled nucleotide analogues (dye labelled nucleotides) commonly used for labeling microarray probes. The inventors engineered a polymerase that can incorporate a higher density of dye-labeled nucleotides into neologistic acid synthesized by that polymerase compared to the wild type polymerase. This results in nucleic acid probes with improved detection of dye labels. The invention also provides a method for improving the sensitivity of PCR, ELISA, FISH, fibre FISH, and microarrays by using a nucleic acid probe that is labeled with dye-labeled nucleotides.

Problems solved by technology

Although in nature high polymerase fidelity is vital for accurate DNA replication it has serious drawbacks for many biotechnological applications.
Specifically, it restricts the use of unnatural or modified nucleotide bases and the applications they enable.
Direct incorporation of dye labelled nucleotides employs polymerase enzymes and is limited by the fact that polymerase enzymes have evolved to preserve a high selectivity for their correct nucleotide substrate.
Although higher fluorescent nucleic acid labelling densities can be attained by indirect labelling methods complete substitution of every reactive nucleotide has not been achieved.
With current indirect DNA labelling methods unable to label 100% of the available positions research efforts have focussed on identifying naturally occurring or mutant DNA polymerase enzymes that are less stringent with regard to their substrate specificity.
Such efforts have met with modest success.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Polymerase
  • Polymerase
  • Polymerase

Examples

Experimental program
Comparison scheme
Effect test

example 1

Cloning of Pfu Polymerase Open Reading Frame into pASK

[0204]The Pfu polymerase open reading frame was amplified by PCR from pETpfu (Lu and Erickson, 1997) using primers 1 and 2. The amplified fragment was restriction digested with NdeI and SalI and ligated into pASKDpo4 (Skerra, 1994) from which the NdeI site at 2421 bp had been eliminated from the pASK vector backbone by site-directed-mutagenesis using primers 3 and 4 thereby creating the expression vector pASKpfu.

[0205]The 3″-5″ exonuclease activity of the Pfu enzyme was disabled by site-directed-mutagenesis of exonuclease domain I (Derbyshire et al., 1995) using primers 5 and 6 and pASKpfu as a template thereby generating pASKpfuexo−.

[0206]The XbaI site present in the Pfu sequence at 1683 bp was removed by site-directed-mutagenesis using primers 7 and 8 and pASKpfuexo− as a template thereby generating pASKpfuexo−1.

[0207]To facilitate library construction by iPCR the BsaI site present in the Pfu sequence at 1636 bp was eliminated ...

example 2

Pfu Protein Expression

[0212]Plasmid constructs or libraries were transformed into E. coli Ace6 or TG1TR and expressed as described (Skerra, 1994). Briefly, transformed Ace6 cells are grown overnight at 37° C. in 2×TY, 0.1 mg / mL ampicillin. For expression overnight cultures were diluted 1:50 in 2×TY 0.1 mg / mL ampicillin, grown to an OD595 of 0.6 at 37° C. and induced for protein expression by the addition of anhydrotetracycline. Protein expression was induced for 6 hours at 37° C.

[0213]Cells were harvested by centrifugation, resuspended in 20 ml (per litre of culture) of buffer A (50 mM Tris pH8.0, 1% glucose, 1 mM EDTA), Buffer B (10 mM Tris pH8.0, 50 mM KCl, 1 mM EDTA, 0.5% NP40) was added to a final volume of 50 ml and cells were lysed for 30 min at 75° C. Debris was pelleted by centrifugation and the NaCl was added to the supernatant to 0.25M final concentration. Then neutralized polyethyleneamine (PEI) was added to a final concentration of 0.1% v / v and precipitate pelleted by ce...

example 3

Pfu Requires Modified CSR Conditions

[0214]Modifications to the previously described CSR protocol were required to enable selection for Pfu variants and especially variants of Pfu able to incorporate labelled nucleotide analogues. CSR conditions described previously (Ghadessy et al., 2001), when performed in 1×Pfu buffer (10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris-Cl pH 8.75, 2 mM MgSO4, 0.1% Triton X-100, 100 μg / mL BSA; Stratagene Ltd), did not enrich for an active Pfu variant (pASKpfuexo-2; see example 1) over an inactive variant (pASKpfuexo-7; see example 1) when present at a 1:100 ratio. The aqueous phase of the emulsion had to be modified to include primers (1 μM), dNTPs (0.1 mM each), RNase (10 μg / mL), glycerol (10% v / v), formamide (1% v / v), DTT (1 mM) in 1×Pfu buffer. Cells expressing the active Pfu variant were mixed at a ratio of 1:100 with cells expressing the inactive Pfu variant and subjected to PCR with primers 40 and 41 either in solution or in emulsion (CSR) under the modi...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The present invention relates to an engineered polymerase with an expanded substrate range characterised in that the polymerase is capable of incorporating an enhanced occurrence of detection agent-labelled nucleotide analogue into nucleic acid synthesised by that engineered polymerase as compared with the wild type polymerase from which it is derived.

Description

FIELD OF THE INVENTION[0001]The present invention relates to DNA polymerases. In particular the invention relates to a method for the generation of DNA polymerases with an enhanced ability to incorporate nucleotide analogues bearing detection agent labelled substituents. Uses of engineered polymerases produced using the methods of the invention are also described.BACKGROUND[0002]Efficient and precise replication of DNA is pivotal to the maintenance, transmission and expression of genetic information. High-fidelity DNA polymerases are the key enzymes responsible for maintaining genome integrity. To avoid the negative consequences of mutations (hereditary and sporadic diseases) high-fidelity DNA polymerases perform an astonishing feat of molecular recognition, selecting the correct nucleotide triphosphate (dNTP) molecule from a pool of very similar substrates and catalysing its incorporation as specified by the template base. DNA synthesis by exonucleolytic proof-reading deficient DNA...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/12C12Q1/68
CPCC12N9/1252C12Q1/6806C12P19/34
Inventor HOLLIGER, PHILIPPRAMSAY, NICOLAJEMTH, ANN-SOFIE
Owner MEDICAL RESEARCH COUNCIL
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com