Single-molecule analysis of DNA replication in Xenopus egg extracts

Hasan Yardimci; Anna B. Loveland; Antoine M. van Oijen; Johannes C. Walter

doi:10.1016/j.ymeth.2012.03.033

Single-molecule analysis of DNA replication in Xenopus egg extracts

Hasan Yardimci, Anna B. Loveland, Antoine M. van Oijen^*, Johannes C. Walter

^*Corresponding author for this work

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Abstract

The recent advent in single-molecule imaging and manipulation methods has made a significant impact on the understanding of molecular mechanisms underlying many essential cellular processes. Single-molecule techniques such as electron microscopy and DNA fiber assays have been employed to study the duplication of genome in eukaryotes. Here, we describe a single-molecule assay that allows replication of DNA attached to the functionalized surface of a microfluidic flow cell in a soluble Xenopus leavis egg extract replication system and subsequent visualization of replication products via fluorescence microscopy. We also explain a method for detection of replication proteins, through fluorescently labeled antibodies, on partially replicated DNA immobilized at both ends to the surface. (c) 2012 Elsevier Inc. All rights reserved.

Original language	English
Pages (from-to)	179-186
Number of pages	8
Journal	Methods
Volume	57
Issue number	2
DOIs	https://doi.org/10.1016/j.ymeth.2012.03.033
Publication status	Published - Jun-2012

Keywords

Single-molecule
DNA replication
Fluorescence microscopy
CELL NUCLEAR ANTIGEN
S-PHASE
ANALYSIS REVEALS
CHROMOSOMAL DNA
POLYMERASE
INITIATION
INCREASES
ASSOCIATION
PROGRESSION
MECHANISM

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title = "Single-molecule analysis of DNA replication in Xenopus egg extracts",

abstract = "The recent advent in single-molecule imaging and manipulation methods has made a significant impact on the understanding of molecular mechanisms underlying many essential cellular processes. Single-molecule techniques such as electron microscopy and DNA fiber assays have been employed to study the duplication of genome in eukaryotes. Here, we describe a single-molecule assay that allows replication of DNA attached to the functionalized surface of a microfluidic flow cell in a soluble Xenopus leavis egg extract replication system and subsequent visualization of replication products via fluorescence microscopy. We also explain a method for detection of replication proteins, through fluorescently labeled antibodies, on partially replicated DNA immobilized at both ends to the surface. (c) 2012 Elsevier Inc. All rights reserved.",

keywords = "Single-molecule, DNA replication, Fluorescence microscopy, CELL NUCLEAR ANTIGEN, S-PHASE, ANALYSIS REVEALS, CHROMOSOMAL DNA, POLYMERASE, INITIATION, INCREASES, ASSOCIATION, PROGRESSION, MECHANISM",

author = "Hasan Yardimci and Loveland, {Anna B.} and {van Oijen}, {Antoine M.} and Walter, {Johannes C.}",

year = "2012",

month = jun,

doi = "10.1016/j.ymeth.2012.03.033",

language = "English",

volume = "57",

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journal = "Methods",

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T1 - Single-molecule analysis of DNA replication in Xenopus egg extracts

AU - Yardimci, Hasan

AU - Loveland, Anna B.

AU - van Oijen, Antoine M.

AU - Walter, Johannes C.

PY - 2012/6

Y1 - 2012/6

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AB - The recent advent in single-molecule imaging and manipulation methods has made a significant impact on the understanding of molecular mechanisms underlying many essential cellular processes. Single-molecule techniques such as electron microscopy and DNA fiber assays have been employed to study the duplication of genome in eukaryotes. Here, we describe a single-molecule assay that allows replication of DNA attached to the functionalized surface of a microfluidic flow cell in a soluble Xenopus leavis egg extract replication system and subsequent visualization of replication products via fluorescence microscopy. We also explain a method for detection of replication proteins, through fluorescently labeled antibodies, on partially replicated DNA immobilized at both ends to the surface. (c) 2012 Elsevier Inc. All rights reserved.

KW - Single-molecule

KW - DNA replication

KW - Fluorescence microscopy

KW - CELL NUCLEAR ANTIGEN

KW - S-PHASE

KW - ANALYSIS REVEALS

KW - CHROMOSOMAL DNA

KW - POLYMERASE

KW - INITIATION

KW - INCREASES

KW - ASSOCIATION

KW - PROGRESSION

KW - MECHANISM

U2 - 10.1016/j.ymeth.2012.03.033

DO - 10.1016/j.ymeth.2012.03.033

M3 - Article

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SN - 1046-2023

VL - 57

SP - 179

EP - 186

JO - Methods

JF - Methods

IS - 2

ER -

Single-molecule analysis of DNA replication in Xenopus egg extracts

Abstract

Keywords

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