Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.
DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.
DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. Probes to single copy regions within euchromatin can be generated through a handful of approaches, including nick-translation or end-labeling of long DNA products1,2 and the incorporation of deoxygenin (DIG)-attached nucleotides and their recognition through a wide variety of group-conjugated antibodies1-3. Visualization of euchromatic sequences in few or single copy number requires the use of either single, large probes with high specific activity or a cocktail of multiple, smaller probes that collectively enhance signal.
In contrast, highly repetitive sequences found in heterochromatin, such as satellite DNAs, are easier targets for DNA ISH because they normally exist as tens to thousands of repeats clustered in single chromosome regions known as blocks. Transposable elements also can be found at high copy numbers at distinct chromosomal loci2. In these cases, single probes with low specific activity can effectively label heterochromatic sequences due to their hybridization at multiple sites. Probes to repetitive sequences can be synthesized commercially as short oligonucleotides (30-50 bp) and chemically conjugated with any of multiple different fluorescent groups. Mapping repetitive sequences within heterochromatin by using genome-sequencing technologies is difficult due to challenges encountered in building scaffolds within highly repetitive satellite blocks4-6,7. Currently, ISH stands as the most effective way of mapping these sequences at the sub-chromosome level. This strategy is important for mapping large numbers of repetitive sequences that are being uncovered by ongoing genome and transcriptome sequencing studies.
The efficiency and ease of mapping repetitive sequences on slide-mounted chromosomes would be greatly enhanced by a simplified protocol for DNA ISH. For example, existing protocols for DNA ISH involve multiple washes of hybridized tissues in formamide solution2,8, thus adding substantially to the required time for mapping sequences and also producing large amounts of chemical waste for this costly reagent. Here we describe a revised DNA ISH method that circumvents the need for formamide washes and utilizes basic laboratory equipment and reagents. This method was originally designed for the rapid mapping of highly repetitive DNA sequences in heterochromatic regions of Drosophila larval neuroblasts by using commercially synthesized oligos that are conjugated with fluorescence dyes. However, this method also works for mapping repetitive sequences by using larger probes synthesized through other means9,10 and across multiple different tissue and chromosome types. Additionally, this method can be used to map euchromatic sequences by using longer or multiple, short probes within the euchromatic sequence of interest.
1. Tissue Dissection and Fixation (60 min)
Figure 1: (A) A 3rd instar Drosophila larva (right), with positions indicated for where to grab the mouth hooks (denoted with *) and 2/3 of the way down the larvae to dissect brains; (left) a schematic of a larva a the same developmental stage, depicting the relative position of the brain within the larval head. (B) The brain and ventral ganglia dissected from a 3rd instar Drosophila larva (right) and a schematic of this tissue (left). (C) 3-day-old Nasonia pupa at the yellow body-red eye stage. (D) A testis pair dissected from a 3 day old Nasonia male pupa (right) with positions indicating where to grab the pupa at the posterior of the abdomen (denoted with *) and midway on the body; (left) schematic depicting male and female wasp pupae; male pupae can be distinguished by wings that do not extend past the saggital profile (black arrow), in contrast to females, which have wings that extend past the profile; the relative position of the testis pair is shown in the male pupa. (E) The apparatus—a string of paperclips—and (F) method used to immerse slides in a vessel of liquid nitrogen. Multiple paperclip strings can be used for simultaneous immersion of multiple slides.
2. In situ Hybridization (30 min on day 1; 1 hr—2.5 hr for long probes—on day 2)
Buffer/Solution Recipes
10x PBS
1x PBT
20x SSC
4x SSCT
0.1x SSC
Hybridization mix (20 µl; modified from 11)
SBT8 (10 ml)
Blocking solution8 (10 ml)
Fixative solution with paraformaldehyde (1 ml)
To demonstrate this method, we hybridized a set of small commercially synthesized oligos that were chemically modified with fluorescent conjugates (Figure 2) and a longer biotinylated probe (made through nick translation of a PCR product; Figure 2B), to chromosomes from several different tissue types (see Table 1). The target sequences included satellite repeats located in pericentromeric (heterochromatic) regions of mitotic chromosomes from D. melanogaster larval neuroblasts (Figure 2A-C), and meiotic chromosomes from pupal N. vitripennis testes (Figure 2D). The hybridizations in each of these cases revealed discrete banding patterns at sub-chromosomal regions. One point worth mentioning is that the probe to the D. melanogaster 359 bp satellite repeat recognizes both a large multi-megabase pair block on the X, and a much smaller region on chromosome 3 (Figure 2A). A longer probe against the Responder satellite also detects repeat blocks consisting of relatively low repeat copy numbers (~700 copies; Figure 2B). Thus, this method allows visualization of very small satellite blocks. To help reveal chromosome structure and aid in chromosome identification, it can be useful to treat the chromosomes with a hypotonic solution (described in step 1.5). The tissues in figure panels 2B and 2C were treated with a hypotonic solution for 5 and 10 min, respectively. Note the separation of sister chromatids is greater with the longer treatment (Figure 2C).
Figure 2: DNA in situ hybridization to (A) larval neuroblast chromosomes from D. melanogaster/D. simulans hybrids; (B) and (C) larval neuroblast chromosomes from D. melanogaster treated with hypotonic solution (see Dissection and Fixation step 1); (D) meiotic chromosomes from pupal testis tissue of the jewel wasp Nasonia vitripennis. In (A), green arrows indicate small regions of AATAT satellite on the D. simulans 4th chromosome, while the green arrowhead points to AATAT on the D. melanogaster X chromosome. Red arrow highlights a small region of 359 bp satellite on the D. melanogaster 3rd chromosome, while the red arrowhead marks 359 bp sequences on the X. All satellites in (A) were hybridized with short, fluorescently labeled oligos. In (B), the green signal corresponds to the AAGAG satellite (synthesized oligo) and the red signal is the 120 bp Responder satellite (a biotinylated probe; marked with red arrows). (C) The same Responder satellite with a short fluorescently labeled oligo (red arrows). In (D), the red arrow marks a satellite that is uniquely located on the Paternal Sex Ratio (PSR) chromosome, which is a non-essential, supernumerary B chromosome found in natural populations of N. vitripennis13, while the green arrow indicates a satellite on one of the normal chromosomes; this hybridization was performed by using short, fluorescently labeled oligos. All probes are detailed in Table 1. Scale bar in panel (A) is 10 µm. Please click here to view a larger version of this figure.
Probe name | Probe type | Oligos | Label |
AAGAG satellite | Oligo | (AAGAG)7 | 5’ 6-FAM (Fluorescein) |
AATAT satellite | Oligo | (AATAT)6 | 5’-Cy3 |
359-bp satellite | Oligo | TTT TCC AAA TTT CGG TCA TCA AAT AAT CAT | 5’-Alexa555 |
PSR satellite | Oligo | TGT AAC TGG AAA AGG AAA ATG TAT TAT TGA | 5’-Alexa555 |
Nasonia autosome satellite | Oligo | AAT TTT GTG AAT TTT GGT GTC TCC ATC | 5’-Alexa633 |
Responder | PCR | F-5’ GGA AAA TCA CCC ATT TTG ATC GC | Biotin-14-dATP |
R-5’ CCG AAT TCA AGT ACC AGA C |
Table 1: Probe types used in Figure 2.
DNA ISH is frequently used to map specific sequences to chromosomes. We have described a simple method for DNA ISH optimized for high copy number, heterochromatic sequences. Rather than using washes in a formamide solution, which is a requirement in other existing DNA ISH protocols, we place tissue-mounted slides directly on a pre-heated block to denature DNA. This method circumvents the use of large amounts of formamide. One critical step for producing crisp hybridization signals is to use freshly made fixation solution; failure to do so (or making new solution with paraformaldehyde that has been open for more than one month) may decrease signal resolution. A limitation of this method is that, like for other DNA ISH protocols, the hybridization temperature may require optimization in order to eliminate spurious hybridization to off-target sequences.
This protocol is sensitive—short, ssDNA probes conjugated with a single fluorophore per molecule can detect blocks of repeats with even relatively low repeat copy numbers (e.g., ~700 Rsp repeats; Figure 2C). Signals to satellite blocks with fewer repeats may be visualized with short ssDNA probes by using a highly sensitive digital camera. Although not shown here, this method is also effective for mapping non-repetitive sequences in euchromatin10. Using long, biotinylated probes provides more flexibility in detecting low copy number sequences as the signal can easily be amplified by repeated treatments of biotinylated anti-avidin and rhodamine-avidin9. To visualize single-copy sequences, multiple probes spanning the sequence may be necessary. Alternatively, bacterial artificial chromosomes (BACs) containing the sequence of interest can be labeled through nick translation or random priming for use as a probe for non-repetitive sequences10. To map sequences on a fine scale, probes targeting different repeats (each with different fluorophores) can be used simultaneously to obtain the position of target sequences relative to other repeats and structural landmarks on the chromosomes. While we use two probes here (Figure 2A, B, D), the number of probes can easily be increased to three10 or more, depending on the number of different microscope fluorescence filters available. This protocol can be extended to other tissues—we have verified that the protocol works well on polytene chromosome squashes from salivary glands. It is conceivable that this method be coupled with immuno-staining of chromatin proteins; however it is recommended that immuno-staining be performed as a first step and followed by re-fixation with paraformaldehyde before DNA ISH in order to prevent antibody loss during DNA ISH.
The authors have nothing to disclose.
We thank Zhaohua Irene Tang in the W. M. Keck Science Department for the use of her epifluorescence microscope and the Werren lab for donating Nasonia for dissections. This work was supported in part by an NIH-NRSA fellowship (5F32GM105317-02) to AML.
Company | |
Materials | |
Poly-L-lysine coated slides (regular slides also can be used) | Sigma Aldrich |
Ultrafine tweezers (5 gauge) | Dumont |
22mmx22mm cover slips, Sigmacote-treated by immersion for 15 seconds, blotting dry, and wiping away all traces of Sigmacote so that cover slip is clear | Fisher |
Sigmacote | Sigma |
Filter paper (75-150mm) | |
Paraffin wax paper | |
Heat block with thermometer | |
Dry incubator | |
Razor blades | |
Humidity chamber (empty pipette tip box or Tupperware, lined with moistened paper towels or Kimwipes) | |
Coplin jars (with slide grooves) | |
Aluminum foil | |
Pasteur pipettes | |
1.5 mL microfuge tubes | |
Nail polish (clear or colored) | |
2-20 μL micropipette and plastic tips | |
20-25 standard metal paperclips linked to form a chain | |
Company/Notes | |
Reagents | |
16% EM grade paraformaldehyde | Electron Microscopy Reagents |
Acetic acid | Sigma |
Liquid nitrogen | |
100% Ethanol, chemical grade | |
Commercially synthesized, fluorescently labeled oligos | |
Long biotinylated probe (e.g. nick translated and biotinylated with BioNick from Invitrogen) | Invitrogen; Alternative steps 2.7.1-2.7.3 |
Rhodamine-Avidin (for detection of long biotinylated probe) | Roche; Alternative steps 2.7.1-2.7.3 |
Hybridization buffer | Recipe below |
4X SSCT (Saline-sodium citrate + Tween) | Recipe below |
0.1X SSC (Saline-sodium citrate) | Recipe below |
Blocking solution | Recipe below |
SBT (SSC, Bovine serum albumin, Tween) | Recipe below |
1X PBT (Phosphate-buffered saline + Tween) | Recipe below |
1X PBS (Phosphate-buffered saline) | |
Hypotonic solution (0.5% sodium citrate in H2O) | |
Formamide | Sigma Aldrich |
Vectashield mounting medium with DAPI | Vector laboratories |