M

 

 

 

 
DNA Combing / DNA Fibre / Fiber spreading for fluorescent in situ hybridization (FISH)

Pat Heslop-Harrison and Trude Schwarzacher
(Updated September 2011 and Jan 2006)
*Department of Biology, University of Leicester, LE1 7RH UK. homepage: www.molcyt.com (this page under techniques section)
e-mail: phh4(a)le.ac.uk

(Techniques based on Michalet, Bensimon et al. 1997 and Jiming Jiang, Madison, Wisconsin)

Reagents - Methods - Troubleshooting/key points - Alternatives - ReferencesIn situ hybridization to spread DNA fibres

Since 2006, we have not used the pulling method so much. Aaron Bensimon now has a company  Genomic Vision (and www.igmm.cnrs.fr which offers both the equipment and treated slides, although we have not tried them. If I were to build another system for pulling, I would use the Lego Technic system which has lots of gears and operates very slowly and smoothly, as well as being very cheap (and more fun) compared to buying motors, gears, supports from lab. supplies companies.

Getting the slides just right to hold the DNA is important. After trying various slide sources and our own coated ones, recently we have be using poly-L-lysine slides from Muto Pure Chemicals, Tokyo, Japan. These we get/bring from Japanese collaborators, and I do not think they are available outside Japan but any lab. there can obtain them. This is no guarantee that another batch bought two years later would still be good in DNA fibre fiber spreading applications..

Pictures of our current type of results are in papers at http://www.le.ac.uk/biology/phh4/openpubs/Dros_junctions_kuhn.pdf (excerpt shown above left) from Heredity and Chromosome Research http://www.le.ac.uk/biology/phh4/HHPubs/buzzsat.pdf (intranet only) (or doi: 10.1007/s10577-007-1195-1 ). The latter gives the protocol we are using now in good detail (based on one from Jiming Jiang in Wisconsin), and we have continued to use this most recently.

This paper gives the protocol for Drosophila, but the same approach has worked for plants. For DNA fibre spreading, high-molecular-weight DNA for in situ hybridization was prepared from fresh Drosophila adults or flies stored at -80C. The protocol used was adapted from Schwarzacher & Heslop-Harrison (2000) and from an online protocol provided by Jiming Jiang formerly at http://www.hort.wisc.edu/jjiang/nuclear_fiber_fish_1.htm (not available 9/2011)..

Approximately 10 to 20 flies were homogenized with a pestle in a microcentrifuge tube containing 500 ul Nuclear Isolation Buffer (NIB: 10 mM Tris-HCl pH 9.5, 10 mM EDTA, 100 mM KCl, 0.5 M sucrose, 4.0 mM spermidine, 1.0 mM spermine, 0.1% mercaptoethanol) and subsequently filtered twice through nylon mesh filters. After addition of 5 ul (micro-litres) of NIB containing 10% Triton X-100 (pre-mixed, final concentration between 0.5% and 1%), the solution was centrifuged (2000 g; 10 min) and the pellet was resuspended in 100-200 ul of 1:1 NIB (without mercaptoethanol) and 100% glycerol. An aliquot of the nuclear suspension (c 10 ul) was transferred to a microcentrifuge tube containing 100 ul NIB, followed by centrifugation (1200 g; 5 min). The pellet of nuclei was subsequently resuspended in 20 ul PBS (10 mM sodium phosphate, pH 7.0, 140 mM NaCl). In one end of a poly-L-lysine slide (Muto Pure Chemicals, Tokyo, Japan), 2 ul of nuclear suspension was added, air-dried for 5Y10 min (but avoiding overdrying) and 8 ul of STE lysis buffer (0.5% w/v SDS, 100 mM Tris-HCl, pH 7.0 and 50 mM EDTA) was added above the nuclei drop. The slides were incubated for 6-10 min (avoiding overdrying) at room temperature and the solution was dragged down the slide with the edge of a coverslip. The slides were air-dried at room temperature, fixed in fresh 3:1 ethanol:acetic acid, air-dried and incubated at 60-C (30 min). The in situ hybridization steps and post-hybridization washes were conducted basically as described for chromosomes, with DNA fibers counterstained with DAPI. The length of single DNA fibers was measured considering that 320 pixels (objective 100) or 180 pixels (objective 63) correspond to 10 mm, which corresponds to 29 kb of the extended DNA fiber (Schwarzacher & Heslop-Harrison 2000). Typically, two slides per species were analyzed and for each slide more than 10 pictures from different slide areas were captured.

Reagents

YOYO-1 DNA stain (Molecular Probes - Invitrogen): dilute in water to 5 nM.

MES (2-[N-Morpholino]ethanesulfonic acid; Sigma M-8250): stock solution of 0.5mM, adjusted to pH 5.5 with KOH. Glue: cyanoacrylate (‘superglue’)

Materials

Cover-slips coated with silane solution (10% (v/v) 3-aminopropyltriethoxysilane (APES) in acetone) 5 minutes. Rinse three times with deionized water (washing step seems crucial to me.)

Teflon reservoir: block of Teflon (that is, PTFE plastic, very slippy, DNA does not stick to it) milled with a slot 4-5 mm wide, length and depth to fit coverslip (18x18 or 22x22 mm). This can be made easily with a drill, and the piece of Teflon costs less than USdollar $1, from e.g. model shop or electronics supply shop (as insulation for power transistors).

Pulling device: another trip to the model shop! Obtain a battery-operated motor, with basic speed control and a gear box allowing speeds of a few revolutions per minute (rpm). Plastic gearboxes with adjustable gears are available (e.g. RS components in UK) for a few dollars. Clamp motor and gears to a retort stand, attach thread to output spindle, pass over the top of the retort stand and onto a clip (either a bulldog-type paper clip or reversed, self-closing forceps) holding the cover-slip in the Teflon reservoir. Extra weight may be needed to ensure smooth movement.

Method

  • Add appropriate amount of DNA (typically 3.75 mg (micro-grams) for a final volume of 2.5ml) to 25 ml of YOYO-1 solution (5nM), and leave at room temperature for 30 minutes.
  • Add then water and MES pH 5.5 to have a final volume of 2.5 ml, and a MES concentration between 50 and 150 mM. The DNA solution can be stored for several weeks at 4oC.
  • Pour the solution in a Teflon reservoir (containing up to 3 ml), then dip a silane treated cover-slip in the solution, and leave for 5 minutes.
  • Pull the cover-slip at a constant speed of 300 mm.s-1. This is the critical step, and seems to need adjustment depending on species and DNA concentration! Glue the cover-slip onto a microscope slide for easy handling (with cyanoacrylate), and bake overnight a 60oC.
  • Dry and examine by fluorescence microscopy using 100x oil immersion lens. It is generally worthwhile to use one or two slides to look at directly by putting the oil drop onto the slide and then examining. These slides cannot be used for in situ hybridization subsequently. You can also put a drop of xylene onto the slide (i.e. coverslip with DNA mounted on the slide) with a coverslip over, and then use immersion oil. After examining, flick off the coverslip.

 Hybridization – see standard in situ protocols, but in brief:

10 minutes in paraformaldehyde

2 washes of 5' in 2x SSC

Alcohol dehydratation (80-100%), and air dry

Add probes, denature 8 minutes at 85C

Hybridize Overnight at 37C

Three washes of 5 minutes in formamide 50% 2x SSC at room temperature

Three washes of 5 minutes in 2x SSC

5 minutes with 0.15g BSA in 4x SSC/0.1% Tween 20

Detection with antiDig and antiBio, one hour at 37C

Three washes of 5 minutes in 4x SSC/Tween

Mounting with Anti-fade, e.g. Citifluor.

Troubleshooting, key points and modifications

The amount of DNA in the solution and the rate of pull are the two variables which are important to control.

The DNA must be very dilute in the pulling solution. If the DNA is much too strong, the stretching will not work and the DNA will clump. If it is a bit too strong, then good stretched fibres can be obtained, but the pictures from the microscope will be impossible to analyse because many fibres will overlap. A good density is one fibre in every 3 microscope fields at x 100.

The rate of pulling must be adjusted to allow the DNA to be pulled gently as is moves through the surface meniscus. If too fast, the DNA will not stick at its end to the slide and will break. Too slow is less of a problem!

The quality of the DNA is quite (not very) important: CTAB purification and being digestable by restriction enzymes should be adequate. amount

Alternative methods

Dissolving DNA in a drop of buffer and running down the sloping slide very slowly.

Embedding DNA in agarose blocks as for PFGE, placing on microscope slide, meltiing and dragging with a coverslip.

These will appear in more detail here; meanwhile see Practical In situ hybridization book.

References