Molecular combing of DNA is an extremely powerful DNA fiber–stretching technique that is often used in DNA replication and genome stability studies. Optimal DNA combing results mainly depend on the quality of the silanized surfaces onto which fibers are stretched. Here we describe an improved method of liquid-phase silanization using trimethoxy-octenylsilane/n-heptane as novel silane/solvent combination. Our simple method produces homogenously modified coverslips in a reproducible manner, but does not require any sophisticated or expensive equipment in comparison to other known silanization protocols. However, DNA fibers were combed onto these coverslips with very good high-density alignment and stayed irreversibly bound onto the surfaces after various denaturing treatments, as required for different immunofluorescent detection of DNA with incorporated modified nucleotides or FISH.

Introduction

Molecular combing of DNA combined with immunofluorescent detection is one of the most efficient techniques for stretching and visualizing single DNA fibers. Since its initial description over 10 years ago (1,2), molecular combing has been used for drawing high-resolution physical maps of genomic regions (3,4) or for the detection of chromosomal rearrangements such as deletions (4) and amplifications (5). It is also widely used to study DNA replication and genome stability. Sites of DNA replication (known as replication eyes) can be directly visualized on combed DNA fibers after proper labeling via incorporation of modified nucleotides into nascent DNA. A number of parameters, such as replication origin density, fork speed, and inter-origin distances, can be obtained from the labeled combed molecules. Studies of replication using DNA combing have been realized in various models, such as the Xenopus in vitro system (6,7,8,9), in mammalian cell lines (10,11,12,13), and in both budding and fission yeasts (14,15). Furthermore, in order to study replication at specific loci, immunofluorescent detection of both hybridized probes and replicated DNA has been successfully carried out on combed fibers (10,11,16). Recently, a number of studies on DNA repair, DNA damage, and intra-S checkpoints using molecular combing have been published (17,18,19,20).

Molecular combing is a simple and reproducible fiber stretching technique (1,2) (Figure 1A). A chemically modified glass coverslip is dipped into a buffered DNA solution. DNA fibers bind to the chemically modified, hydro-phobic surface by one or both of their extremities in a pH-dependent manner. When the coverslip is pulled out with a slow and constant speed (v = 300 µm s⁻¹) (4), the receding meniscus stretches the anchored DNA molecules onto the coverslip as it applies a constant perpendicular force on them. This rapid process results in irreversibly fixed DNA fibers and has the major advantage—in comparison to other known fiber-stretching techniques—that DNA fibers are aligned in parallel all over the surface. The stretching factor is constant (1 µm ∼ 2 kb) (1,2), so that internal size standards are not necessary once calculated under the same lab conditions.

Figure 1. DNA combing on various surfaces. (Click to enlarge)

Most of the current applications of molecular combing rely on good density and alignment of the combed molecules. Both depend on the quality of the coverslip surface modification, which has to be homogeneous to support DNA binding on its entire surface. Different surface modifications have been studied, but the chemical surface modification ensuring a best-possible stretching of the DNA fiber is the coating of the surface by an octenyl carbon chain (1,21), achieved by a silanization reaction (22). Only this surface modification assures that DNA fibers are irreversibly fixed and are not lost during treatments, especially from denaturation of combed double-stranded DNA by chemicals or heat, which is necessary for FISH and detection of replication eyes.

The key point for a wider utilization of the DNA combing technique is the availability of high-quality hydrophobic silanized coverslips, which, so far, are not commercially available and need to be homemade. Several silanization methods for surfaces exist, but the most adapted and reproducible coverslips for molecular combing have been obtained, until now, by gas-phase silanization methods (1,21). However, gas-phase silanization requires controlled anhydrous conditions in specialized incubators, which are difficult to use in common biology laboratories. Here, we describe an improved and reproducible liquid-phase silanization method using a novel solvent/silane combination that is practicable in just about any laboratory. We further validate and characterize the silanized surfaces for molecular combing of DNA and its applications in molecular biology.

Materials and Methods

Chemicals and products

All chemical products need to be of the highest quality. Acetone, methanol, and chloroform were purchased from VWR France (Fontenay-sur-Bois, France). All other chemicals and (7-octen-1-yl) trimethoxysilane (Catalog no. 452815) were purchased from Sigma-Aldrich France (Saint Quentin Fallavier, France). We used 20 × 20 mm or 22 × 22 mm glass cover-slips purchased from Esco (Portsmouth, VA, USA). Coverslips were generally silanized in a batch of no more than 25 units.

Combing apparatus

Our combing device was purchased from the Pasteur Institute (Paris, France), which no longer sells the machine commercially. Motorized platforms combined with adapted clamps can be used alternatively.