Quantifying single gene copy number by measuring fluorescent probe lengths on combed genomic DNA

Herrick, John H.; Michalet, Xavier; Conti, Chiara; Schurra, Catherine; Bensimon, Aaron

doi:10.1073/pnas.97.1.222

Cited by 41 publications

(32 citation statements)

References 26 publications

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“…Physical mapping of the human genome by in situ hybridization was an important application of molecular combing (18,19). Here we show that we can extend the combing process to image protein-DNA interactions.…”

Section: Discussionmentioning

confidence: 99%

“…By contrast, molecular combing is of particular interest because it allows direct observation of a large array of immobilized and aligned DNA by fluorescence microscopy. This latter technique was successfully applied to the identification of genomic DNA regions using fluorescent in situ hybridization (18,19), to optical mapping (20, 21), or to the identification of replication origins (22, 23). Nevertheless, this method presents several drawbacks.…”

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confidence: 99%

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Observation by fluorescence microscopy of transcription on single combed DNA

Gueroui

Place²,

Freyssingeas³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

105

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Molecular combing is a powerful procedure for aligning a large array of DNA molecules onto a surface. This technique usually leads to an overstretching of about 150% of the molecules' contour length. By changing the magnitude of capillary forces during the combing process, we were able to reduce the relative extension of the DNA molecules. Thus we achieved combing of T7 DNA with an extension close to its molecule contour length. We checked the ability of combed DNA to interact with DNA binding proteins. Using the T7 bacteriophage transcription system, we investigated the transcription activity of RNA polymerase on combed DNA by direct visualization of newly synthesized fluorescent RNAs. Our experiments show that no transcription activity occurs on overstretched DNA molecules, whereas we observe a transcription activity for nonoverstretched molecules. This activity is observed both in multiple initiation experiments and for one immobilized T7 RNA polymerase per promoter. These results open possibilities for the study of single enzyme actions on combed DNA by optical methods. T ranscription is a fundamental process in gene expression that allows the regulation of cellular adaptation and differentiation. This function is carried out by RNA polymerases (RNAPs) that produce an RNA copy of a given DNA strand (1). The interaction between the DNA and the RNAP is complex because RNAP must recognize a promoter, a sequence-specific region of doublestranded DNA before polymerization. After isomerization of the nucleoprotein complex, resulting in local melting of the double helix, the enzyme transcribes DNA into RNA following the doublestranded DNA until it reaches a terminator (2). Motion is therefore part of the intrinsic activity of RNAP (3). Having the possibility to follow and visualize the movement of an RNAP on the DNA template will open a new area of investigation for understanding the mechanisms of transcription and its regulation.Our goal is to detect the activity of RNAP along a DNA molecule during the transcription process. Several groups have reported single molecule investigation of the transcription process (4-9). Because we use fluorescence microscopy techniques, we cannot use DNA in its normal aqueous solution state, which is a Brownian fluctuating coil. Therefore the first steps we have to perform are to stretch the DNA molecule to avoid conformational fluctuations and to hinder its Brownian motion. Several techniques permit researchers to obtain both immobilized and stretched DNA molecules. The techniques developed so far are micromanipulation with optical or magnetic tweezers (10-14), elongation in a flow (15) or in an electric field (16), and molecular combing (17). Although micromanipulation has provided much insight it has an inherent disadvantage because the observation is limited to one molecule at a time and large statistics are difficult to obtain. By contrast, molecular combing is of particular interest because it allows direct observation of a large array of immobilized and aligned DNA by fluores...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Observation by fluorescence microscopy of transcription on single combed DNA

Gueroui

Place²,

Freyssingeas³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

105

View full text Add to dashboard Cite

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“…These properties allowed the application of molecular combing to genetic diagnostics, such as quantifying the length of microdeletions 12 and the copy number in replicated oncogenes. 13 The technique of molecular combing has also been used to study the activity of DNA polymerase 14 and to provide a template for the fabrication of palladium nanowires. 15 While several methods exist for extending DNA molecules on a surface, [16][17][18][19][20] we limit our discussion to the growing number of variants of molecular combing.…”

mentioning

confidence: 99%

Combing of Molecules in Microchannels (COMMIC): A Method for Micropatterning and Orienting Stretched Molecules of DNA on a Surface

Petit

Carbeck

2003

Nano Lett.

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This paper presents a new method for creating microscopic patterns of stretched and oriented molecules of DNA on a surface. Combing of molecules in microchannels (COMMIC), a process by which molecules are deposited and stretched onto a surface by the passage of an air−water interface, creates these patterns. This approach demonstrates that the direction of stretching of the molecules is always perpendicular to the air−water interface; the shape and motion of this interface serve as an effective local field directing the chains dynamically as they are stretched onto the surface. The geometry of the microchannel directs the placement of the DNA molecules, while the geometry of the air− water interface directs the local orientation and curvature of the molecules. This ability to control both the placement and orientation of chains has implication for the use of COMMIC in genetic analysis and in the bottom-up approach to nanofabrication.

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“…Depletion of And-1 Reduces Both the Velocity and Density of Replication Forks-To further characterize the role of And-1 in DNA replication, we examined the progression of replication forks (fork velocity) and the number of forks per length of DNA (fork density) in siGl2-and siAnd-1-treated cells using molecular combing (34,40,41). Cells transfected with either siGl2 or siAnd-1 were labeled with 100 M IdU for 20 min and then 100 M CldU for 20 min before being harvested at 36 h after transfection.…”

Section: And-1 Regulates MCM Proteinsmentioning

confidence: 99%

The Involvement of Acidic Nucleoplasmic DNA-binding Protein (And-1) in the Regulation of Prereplicative Complex (pre-RC) Assembly in Human Cells

Xiao

Renty

et al. 2012

Journal of Biological Chemistry

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Background:The assembly of a replicative helicase MCM2-7 onto replication origins is essential for initiation of DNA replication. Results: And-1 facilitates assembly of MCM2-7 onto replication origins. Conclusion: And-1 is proposed to be a critical regulator of prereplicative complex assembly in human cells. Significance: These studies reveal a novel role for human And-1 in the licensing of replication origins.

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Quantifying single gene copy number by measuring fluorescent probe lengths on combed genomic DNA

Cited by 41 publications

References 26 publications

Observation by fluorescence microscopy of transcription on single combed DNA

Observation by fluorescence microscopy of transcription on single combed DNA

Combing of Molecules in Microchannels (COMMIC): A Method for Micropatterning and Orienting Stretched Molecules of DNA on a Surface

The Involvement of Acidic Nucleoplasmic DNA-binding Protein (And-1) in the Regulation of Prereplicative Complex (pre-RC) Assembly in Human Cells

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