Dynamic molecular combing: Stretching the whole human genome for high- resolution studies

Science

Volumn 277, Issue 5331, 1997, Pages 1518-1523

  Michalet, Xavier ^a   Ekong, Rosemary ^a   Fougerousse, Françoise ^a   Rousseaux, Sophie ^a   Schurra, Catherine ^a   Hornigold, Nick ^a   Van Slegtenhorst, Marjon ^a   Wolfe, Jonathan ^a   Povey, Sue ^a   Beckmann, Jacques S ^a   Bensimon, Aaron ^a  

^a NONE

Author keywords

[No Author keywords available]

Indexed keywords

DNA; FUNGAL DNA;

ARTICLE; DNA PROBE; GENOME; HUMAN; MOLECULAR DYNAMICS; MOLECULAR GENETICS; PRIORITY JOURNAL; REPRODUCIBILITY; YEAST ARTIFICIAL CHROMOSOME;

CALPAIN; CHROMOSOME MAPPING; CHROMOSOMES, ARTIFICIAL, YEAST; CLONING, MOLECULAR; COSMIDS; DNA PROBES; ELECTROPHORESIS, GEL, PULSED-FIELD; GENETIC TECHNIQUES; GENOME, FUNGAL; GENOME, HUMAN; HUMANS; IN SITU HYBRIDIZATION, FLUORESCENCE; ISOENZYMES; MUSCLE PROTEINS; MUSCULAR DYSTROPHIES; MUTATION; PROTEINS; REPRESSOR PROTEINS; REPRODUCIBILITY OF RESULTS; SEQUENCE DELETION; SILANES; TUBEROUS SCLEROSIS; TUMOR SUPPRESSOR PROTEINS;

EID: 13044316665     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.277.5331.1518     Document Type: Article

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27. Shorter incubation times (30 s) of the slides in the DNA solution have also been used successfully, implying that the kinetics of DNA binding is probably very fast, with a rapid saturation of all accessible binding sites. The quality of combing does not depend on the speed at which cover slips are pulled out of the DNA solution [speeds of up to 500 μm/s have been used (10)].
28. This parameter is constant for each batch of treated surfaces. For unexplained reasons, a few batches with a stretching factor of 2.3 kb/μm have sometimes also been observed. Hence, systematic measurements of λ-DNA molecules were performed on a few surfaces per batch (each batch containing 72 surfaces). Surface treatment inhomogeneities on each cover slip may occur and lead to local variations in the surface density of combed molecules, or in the stretching factor. However, these inhomogeneities have no effect on the final results obtained on a large number of measurements, as shown by their small standard deviation (see text). Residual surface defects may also result in transient pinning of the meniscus, leading to a local deformation of the normally straight interface. It can be detected as a local departure from parallelism, but has no effect on the stretching factor. The validity of this factor for a wide range of DNA sizes was shown by measurement of molecules ranging from ∼50 kb to ∼250 kb (11) and by redundant measurements of cosmid distances (21).
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31. Probe labeling: all probes consisted of genomic DNA cloned in cosmids. Cosmids were grown and DNA extracted with a scaled-up alkaline lysis method (3). Each probe (1 μg) was labeled by random priming with either biotin-14-deoxycytosine triphosphate (Bioprime kit, Gibco-BRL) or digoxigenin-11-deoxyuridine triphosphate (DIG, Boehringer) according to manufacturer's instructions. Ethanol-precipitated probes were resuspended in 50 μl of 10 mM tris/1 mM EDTA (pH 8) and stored at -20°C. Concentrations were estimated by densitometry on a 0.6% agarose gel. Probes labeled by nick-translation have also been used. We again ethanol precipitated 500 to 600 ng of each labeled probe in the presence of 5× excess human COT-1 DNA (except when hybridizing on yeast DNA, for which no COT-1 was used) and 20 μg of herring sperm DNA at -70°C for 1 hour. After centrifugation, the pellet was dried for 10 min, then resuspended in 10 μl of hybridization mixture [50% formamide, 10% dextran sulfate, 2× saline sodium citrate (SSC), and 1% Tween-20]. Resuspended probes were denatured (5 min in boiling water), snap-cooled, and left on ice until needed. Slides: Combed DNA cover slips mounted on microscope slides were equilibrated to RT, dehydrated through ethanol series (70%, 90%, 100%) three times of 3 min each, air-dried, denatured in 50% formamide/ 2× SSC (pH 7.0) at 75°C for 2 min, then quenched in ice-cold ethanol series and air dried. Hybridization: Denatured probes were incubated with combed DNA at 37°C under an ordinary glass cover slip, with the edges sealed with rubber cement, for at least 16 hours. Detection: All slides were washed at RT on a shaker as follows: three washes of 5 min in 50% formamide/2× SSC (pH 7) and three washes of 5 min in 2× SSC. We applied 50 μl of 1.5% blocking solution (Boehringer) on each DNA cover slip and incubated them at 37°C for 30 min in a moist chamber. Biotinylated and DIG-labeled probes were detected with Texas red and FITC, respectively, using five successive layers of antibodies as follows: (i) avidin-Texas red 1/50 (Vector Labs) + anti-DIG-FITC (mouse) 1/50 (Jackson ImmunoResearch Labs); (ii) biotinylated anti-avidin 1/100 (Vector Labs) + anti-mouse-FITC (donkey) 1/50 (Jackson ImmunoResearch); (iii) avidin-Texas red 1/50 + anti-rabbit (mouse) 1/50 (Jackson ImmunoResearch); (iv) biotinylated anti-avidin 1/100 + F1 (anti-FITC; Cambio) 1/400; and (v) avidin-Texas red 1/50 + F2 (anti-anti-FITC; Cambio) 1/100. The last two layers were necessary to increase the intensity of the signals. All antibody incubations were at 37°C; the first three were for 30 min each and the last two for 20 min each. All antibody washes were for three times of 5 min at RT using 4× SSC/0.05% Tween-20. A final wash in PBS was performed, and the slides were drained and mounted in Vectashield (Vector Labs) without counterstain.
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38. Redundant measurements consist of measuring the distance between cosmid A and B, the distance between B and C, and the distance between A and C in three different hybridization assays. Any stretching inhomogeneity would give different results for the distance between A and C, as obtained from the sum of the first two hybridizations and from the direct measurement of the third one. A perfect agreement was observed in all independent tests (over a 200-kb region), which allowed us to convert mean sizes measured in micrometers to sizes measured in kilobases, without needing to refer to an internal control. Direct measurements have standard deviations of 6 to 11%. The gap size was measured indirectly from two measurements, which led to a larger standard deviation (21).
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46. This yield of about 50 to 100 pairs of hybridized cosmids is in agreement with the rough estimate of DNA density of, at most, a few hundred combed genomes per slide (14). The total number of genomes as estimated, also including the incomplete and isolated signals, is indeed much closer to the one estimated from combing density, thus showing a satisfactory hybridization efficiency (R. Ekong and X. Michalet, data not shown; J. Herrick et al., in preparation).
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62. X.M. benefited from a Pasteur-Weizmann grant, S.R. from a Wellcome Trust grant, and M.V.S. from support by the Dutch Organisation for Scientific Research. We thank A. Chiffaudel for technical help in the development of the combing apparatus, J. Sampson and P. Harris for providing us with TSC2 probes and patient cell lines, and J. Nahmias for unpublished data on the TSC1 contig. Supported in part by the Association Française contre les Myopathies. Dedicated to A. Ullmann for constant support throughout this project.