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Estimation of Purity of Flow-Sorted Chromosomes

Estimation of Purity of Sorted Chromosomes Using FISH

This protocol describes a procedure that allows precise estimation of the purity of a sorted fraction (Kubalakova et al. 2000). The procedure is based on specific fluorescent labelling of repetitive DNA sequences that show characteristic pattern of distribution among the chromosomes. The sequences are labelled using hybridization of specific probes labelled with biotin or digoxigenin and detected with avidin, streptavidin or anti-digoxigenin antibody conjugated with fluorochromes. The chromosomes are evaluated using a fluorescence microscope.

I. Sort Chromosomes
1.Pipet 10 µl of P5 buffer containing 5% sucrose (w/v) onto a clean microscope slide.
2.Immediately sort 1000 chromosomes into the drop.
II. Perform FISH Reaction
1.Pipette 10µl P5 Buffer onto a clean microscopic slide and immediately sort 1000 chromosomes into the drop
2.Air-dry the slide and store at room temperature
3.Add 25µl Hybridization Mix to the area containing flow-sorted chromosomes, cover with a glass coverslip and seal up with rubber cement.
4.Denature for 45 sec at 80°C by placing the slide on a temperature controlled hot plate.
5.Transfer the slide to a wet chamber and incubate at 37°C overnight.
6.Remove carefully the coverslip and wash in 2 × SSC for 10 min at 42°C
7.Perform a stringent wash in 0.1 × SSC for 5 min at 42°C.
8.Wash again 10 min in 2 × SSC and next 10 min in 2 × SSC decrease gradually temperature to room temperature.
9.Wash in 4 × SSC for 10 min at room temperature.
10.Apply 60µl 1% Blocking Buffer onto the slide, cover with parafilm and incubate for 10 min in at room temperature. Repeat this step two times.
11.Apply fluorescently labeled antibody and/or fluorescently labeled avidin in 1% Blocking Buffer for 1 hour at 37°C
12.Wash the slide three times in 4 × SSC for 5 min at 40°C.
13.Mount the slide with Vectashield containing DAPI.
14.Use fluoresce microscope to identify sorted chromosomes and determine the presence of contaminating chromosomes by evaluating at least 100 chromosomes on three different slides.

References 

Kubaláková, M., Valárik, M., Bartoš, J., Vrána, J., Číhalíková, J., Molnár-Láng, M., Doležel, J.: Analysis and sorting of rye (Secale cereale L.) chromosomes using flow cytometry. - Genome 46: 893-905, 2003. 
Doležel, J., Kubaláková, M., Bartoš, J., Macas, J.: Flow cytogenetics and plant genome mapping. - Chrom. Res. 12: 77-91, 2004.
Šafář, J., Bartoš, J., Janda, J., Bellec, A., Kubaláková, M., Valárik, M., Pateyron, S., Weiserová, J., Tušková, R., Číhalíková, J., Vrána, J., Šimková, H., Faivre-Rampant, P., Sourdille, P., Caboche, M., Bernard, M., Doležel, J., Chalhoub, B.: Dissecting large and complex genomes: flow sorting and BAC cloning of individual chromosomes from bread wheat. – Plant J. 39: 960-968, 2004.
Kubaláková, M., Kovářová, P., Suchánková, P., Číhalíková, J., Bartoš, J., Lucretti, S., Watanabe, N., Kianian, S.F., Doležel, J.: Chromosome sorting in tetraploid wheat and its potential for genome analysis. – Genetics, 170: 823-829, 2005.

Estimation of Purity of Sorted Chrmosomes Using PRINS

This protocol describes a procedure that allows precise estimation of the purity of a sorted fraction (Kubalakova et al. 2000). The procedure is based on specific fluorescent labelling of repetitive DNA sequences that show characteristic pattern of distribution among the chromosomes. The sequences are labelled using a primed in situ labelling (PRINS) and the chromosomes are evaluated using a fluorescence microscope.

I. Sort Chromosomes

  1. Pipet 15 µl of LB01 buffer (for barley) or PRINS buffer (for legumes and wheat) containing 5% sucrose (w/v) onto a clean microscope slide.
  2. Immediately sort 1000 chromosomes into the drop.
  3. Air-dry in an aseptic box for about 1h.

II. Perform PRINS Reaction

  1. Stick 'Frame-Seal' incubation chamber to the slide over the specimen area.
  2. Pipet 25-µl of PRINS reaction mix into the frame and place a polyester coverslip over the frame.
  3. Run the PRINS reaction using the following PCR cycles:
    1 cycle: 5 min 94°C (denaturation)
    5 min 55°C (annealing)
    10 min 72°C (extension)
    8 cycles: 1 min 91°C (denaturation)
    1 min 55°C (annealing)
    3 min 72°C (extension)
    1 cycle: 1 min 91°C (denaturation)
    5 min 55°C (annealing)
    10 min 72°C (extension)
  4. Remove the cover, add 100 µl of the stop buffer, and incubate for 2 min at 70°C.
  5. Remove the stop buffer and transfer the slide to a 10-cm petri dish.
  6. Add 70 µl wash buffer and incubate at room temperature for 5 min. Repeat the washing step twice.

III. Examine Slides

  1. Add 70 µl of wash buffer containing DAPI (0.2 µg/ml) to counterstain the chromosomes.
  2. Drain excess fluid, but not dry.
  3. Add 8 µl of Vectashield antifade solution and cover with a glass coverslip.
  4. Gently squeeze out excess solution and seal with rubber cement.
  5. Examine the slide with fluorescence microscope.

Notes

The drops are dried within 1 h. However, the PRINS reaction should be performed next day after overnight incubation at room temperature. 
The actual conditions for the PRINS reaction (i.e., denaturation and annealing temperature) must be optimised by for given species and primer pair. 
During slide observation, use a DAPI filter first to localise sorted chromosomes. Avoid prolonged exposure to excitation light that fades rapidly both DAPI and fluorescein.

References 

Kubalakova M, Lysak MA, Vrana J, Simkova H, Cihalikova J, Dolezel J. Rapid identification and determination of purity of flow-sorted plant chromosomes using C-PRINS. Cytometry 41: 102 - 108 (2000) Abstract PDF

Estimation of Purity of Sorted Chrmosomes Using PCR with Chromosome-Specific Primers

I. Sort Chromosomes

  1. Prepare PCR tubes containing 19 µl of sterile deionized H2O (final volume after sorting will be approximately 20 µl).
  2. Sort 500 chromosomes into each tube.
  3. Freeze the tube and store it at -20°C.

II. Perform PCR Reaction

  1. Thaw a chromosome fraction.
  2. Add 30 µl of PCR premix, vortex and spin briefly.
  3. Perform PCR amplification using the following cycles:
    1 cycle: 5 min denaturation at 94°C
    35-40 cycles: 30 sec denaturation at 94°C
    1 min annealing at the optimised temperature
    2-3 min extension at 72°C
    1 cycle: 10 min extension at 72°C
  4. Hold the samples at 4°C.

III. Analyze PCR Products

  1. Take equal amounts of PCR products (5 - 10 µl) from each tube and add 1 - 2 µl of loading buffer.
  2. Load the samples onto the agarose gel bathed in TAE buffer.
  3. Load DNA molecular weight markers.
  4. Run electrophoresis; stop when bromophenol blue reaches a point 3 cm from the edge of the gel.
  5. Stain the gel with ethidium bromide working solution (0.5 mg/ml).
  6. Photograph the gel and analyse the presence of products in individual lanes.

Notes

This protocol was optimised for RFLP-derived markers in barley. For other types of markers and plant species, slight modification might be necessary. 
It is important to freeze the tubes even if the reaction is to be performed on the same day of sorting. 
Annealing temperature must be optimised by for given primer pair and the template. 
The electrophoresis should be run at constant voltage of 4 - 5 V/cm.

References

Lysak MA, Cihalikova J, Kubalakova M, Simkova H, Kunzel G, Dolezel J. Flow karyotyping and sorting of mitotic chromosomes of barley (Hordeum vulgare L.). Chromosome Research 7: 431 - 444 (1999)