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Genetics / Genomics JoVE (Open Access) Citable · DOI

2D and 3D Chromosome Painting in Malaria Mosquitoes

DOI: 10.3791/51173-v
What you'll learn
  • ✓ Isolate and amplify specific chromosomal regions from polytene chromosomes using laser capture microdissection
  • ✓ Prepare fluorescent FISH probes via whole genome amplification for chromosome visualization
  • ✓ Perform 2D and 3D fluorescent in situ hybridization to map chromosome organization and karyotype evolution
Protocol

Chromosome painting is a useful method for studying organization of the cell nucleus and evolution of the karyotype. Here, we demonstrate an approach to isolate and amplify specific regions of interest from single polytene chromosomes that are subsequently used for two- and three-dimensional fluorescent in situ hybridization (FISH).

Difficulty
advanced
Total time
~3–5 days (including probe preparation, hybridization, and imaging)
Model organism
Anopheles gambiae (malaria mosquito)
Biosafety
BSL-1

Steps

1
Isolate DNA from polytene chromosomes by laser microdissection

Extract specific chromosomal regions of interest from polytene chromosomes using laser capture microdissection technology. This isolates target DNA segments for downstream amplification and probe synthesis.

▶ 01:06
2
Amplify isolated chromosomal DNA via whole genome amplification

Use whole genome amplification (WGA) kits to amplify the microdissected chromosomal DNA to sufficient quantities for probe labeling and FISH experiments.

▶ 02:07
3
Perform 3D FISH on whole-mount ovarian tissue samples

Apply three-dimensional fluorescent in situ hybridization to whole-mount ovarian tissue using labeled probes to visualize chromosome organization and spatial distribution in intact tissues.

▶ 03:59
4
Assess probe yield and FISH labeling efficiency

Evaluate whole genome amplification kit yields and quantify fluorescent labeling of probes to confirm probe quality and readiness for hybridization experiments.

▶ 07:33
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