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Polymerase-template complexes

a polymerase and template complex technology, applied in the direction of enzymology, enzyme stabilisation, transferases, etc., can solve the problems of unable to optimize specific variables (conditions) that optimize certain sequencing reactions, such as nanopore based sequencing, and achieve the effect of increasing the processivity of the template-polymerase complex and the processivity of the polymerase template complex formed in high-temperature solution

Inactive Publication Date: 2017-09-21
GENIA TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patent describes methods and compositions for optimizing sequencing reactions, particularly nanopore-based sequencing. The methods involve using high salt concentration to enhance the sequencing reaction. The polymerase-template complex is prepared by contacting a polymerase with a polynucleotide template in a solution containing a high concentration of salt and being free of nucleotides. The resulting complex has greater processivity, meaning it can better process the template. The methods also involve using low nucleotide concentrations and high temperatures to further enhance the sequencing reaction. These methods and compositions can be used to improve the accuracy and efficiency of sequencing.

Problems solved by technology

A crucial obstacle to the success of nanopores as a reliable DNA analysis tool is the processivity or average read length.
Due to the numerous varying conditions at which the sequencing reaction can be ran, however, the specific variables (conditions) that optimize certain sequencing reactions, such as nanopore based sequencing, have remained largely elusive.

Method used

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Examples

Experimental program
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example embodiments

[0080]In certain example embodiments, the present disclosure provides methods and compositions for enhancing the processivity of the polymerase during template-dependent polynucleotide synthesis in the presence of a high concentration of salt. In other example embodiments, the present disclosure provides methods and compositions for enhancing the processivity of the polymerase during template-dependent polynucleotide synthesis in the presence of low nucleotide concentrations and high temperatures. The methods and compositions provided are applicable to methods of template-dependent DNA synthesis, including DNA amplification and sequencing. Sequencing methods include sequencing-by-synthesis of single polynucleotide molecules, such as nanopore sequencing of single DNA molecules.

[0081]As illustrated in FIG. 1, the processivity of a polymerase, such as a DNA polymerase, is directly related to the formation of the polymerase-template complex and the incorporation of dNTP by the enzyme. U...

example 1

Mutagenesis

Pol6 Mutants

[0177]DNA of SEQ ID NO: 3 encoding the WT-Pol6 (SEQ ID NO: 2) was purchased from a commercial source (DNA 2.0, Menlo Park, Calif.). The sequence was verified by sequencing.

[0178]Site directed mutagenesis was performed to mutate one or more amino acids of the putative nucleotide / DNA binding site of parental variant Pol6-44-X1 (SEQ ID NO:4). Pol6-44-X1 was derived from wild-type Pol6 to comprise the following substitutions: S366A T529M A547F D44A (SEQ ID NO:4). Pol6-67-X2 was derived from wild-type Pol6 to comprise the following mutations: S366A T529M A547F N545L Y225L D657R Y242A (see SEQ ID NO: 14).

[0179]The Pol6 variants like 44-X1 were expressed as a fusion protein having an N-terminal His-tag (see underlined sequence in SEQ ID NO:4) and SpyCatcher domain (bolded italic sequence in SEQ ID NO:4).

Mutagenesis Protocol

[0180]The primers for each mutagenesis reaction were designed using the NEB base changer protocol and ordered in 96-well plate format from IDT.

[01...

example 2

n and Purification

[0185]Variants of the parental polymerase Pol6-44-X1 (SEQ ID NO:4), and the Pol6-67-X2 (SEQ ID NO: 14) were expressed and purified using a high throughput method as follows.

[0186]DNA encoding variants in expression plasmid pD441 vector were transformed into competent E. coli, and glycerol stocks of the transformed cells were made. Starting from a tiny pick of the glycerol stock, grow 1 ml starter culture in LB with 0.2% Glucose and 100 μg / ml Kanamycin for approximately 8 hrs. Transfer 25 μl of log phase starter culture into 1 ml of expression media (Terrific Broth (TB) autoinduction media supplemented with 0.2% glucose, 50 mM Potassium Phosphate, 5 mM MgCl2 and 100 μg / ml Kanamycin) in 96-deep well plates. The plates were incubated with shaking at 250-300 rpm for 36-40 hrs at 28° C.

[0187]Cells were then harvested via centrifugation at 3200×g for 30 minutes at 4° C. The media was decanted off and the cell pellet resuspended in 200 μl pre-chilled lysis buffer (20 mM P...

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Abstract

The present disclosure provides methods and compositions for enhancing the processivity of a polymerase in catalyzing template-dependent DNA synthesis in high concentrations of salt. Also disclosed are methods and compositions for enhancing the assembly of polymerase-template complex compatible with active DNA synthesis in the presence of low levels of nucleotides and at a high temperature, such as temperatures at or near the melting temperature of the polymerase.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]The present application claims the benefit of U.S. Provisional Patent Application No. 62 / 406,431 filed Oct. 11, 2016 and U.S. Provisional Patent Application No. 62 / 301,607 filed Feb. 29, 2016, the disclosures of which are each incorporated herein by reference in their entirety.SEQUENCE LISTING[0002]The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 1, 2017, is named 04338_531US1_SL.TXT and is 60,572 bytes in size.TECHNICAL FIELD[0003]The present disclosure relates generally to methods and compositions for improving DNA sequencing processivity, and more particularly to enhancing sequencing yield in a DNA sequencing reaction via adjustment of temperature, nucleotide concentration, and / or polymerase concentration.BACKGROUND[0004]Nanopores have recently emerged as a label-free platform for interrog...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68
CPCC12Q1/6869C12Y207/07007C12Q1/6806C12N9/1252C12N9/96C12Q2565/631
Inventor AYER, ARUNASARVABHOWMAN, PREETHISCHWAB, CHARLES
Owner GENIA TECH
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