Polytene Chromosomes
Large chromosomes called polytenes are seen in the salivary glands of insects. They were found by Balbiani in 1881. Polytene Chromosomes were found in the salivary glands, they are often referred to as salivary gland chromosomes. They are referred to as polytene because of their large number of chromonemata.
Drosophila melanogaster larvae’s salivary glands contain polytene chromosomes, which are an invaluable resource for researching gene expression and genomic structure. Through in-situ hybridization (ISH), their distinct structure—which is characterized by enormous amplification and banding patterns—allows for the accurate localization of certain DNA sequences. With the use of this method, scientists may see where genes, regulatory elements, and other genomic characteristics are located in relation to the complete chromosome.
A thorough procedure for getting Drosophila polytene chromosomes ready for ISH will be provided by this comprehensive guide. It includes every necessary process, including squashing and fixing chromosomes and collecting and dissecting larvae. We will also go over typical problems and solutions to make sure your experiments turn out as best they can.
Table of Contents
Larval Gathering and Arrangement
Choosing the Larval Stage
Choose larvae that are well segmented and big in size during the third instar stage. For ISH, this stage offers the ideal chromosomal size and banding resolution
Larval Gathering
Gather the larvae from a healthy Drosophila culture and gently place them in a petri dish with a little quantity of fresh food.
To guarantee ideal growth and stop larval cannibalism, avoid overcrowding.
Larvae from a culture that is actively producing eggs, a sign of a vigorous and healthy population, will produce the highest yield.
Dissection of Larva
Give the larvae a short period of anesthesia with a mild anesthetic, such as CO2 or ether.
After the larvae have been put to sleep, transfer them to a dissection dish with a drop of phosphate-buffered saline (PBS).
The salivary glands are two white, elongated organs that are placed on each side of the skull. Carefully dissect them with fine forceps.
After removing the salivary glands from the larval body, put the glands on a freshly prepared PBS-filled petri plate.
Assembly of the Chromosome
Treatment Hypotonic
The salivary glands should be transferred to a tiny tube filled with a hypotonic solution, such as 0.5% sodium citrate.
Give the glands five to ten minutes at room temperature to incubate in the hypotonic solution. This therapy causes the cells to expand and makes it easier for chromosomes to disseminate.
Squashing
Get the cover slips and glass slides ready.
Place one salivary gland on a sterile slide.
To release the chromosomes, gently puncture the gland using a dissecting needle.
Put a drop of the fixative solution on the slide (e.g., 45% acetic acid).
Use a cover slip to gently massage the gland in order to compress the chromosomes.
Let the slide air dry entirely.
Fixation
Fix the squashed Polytene chromosomes by letting the slide sit at room temperature for ten to fifteen minutes in a fixative solution (such as 4% paraformaldehyde in PBS).
Using PBS, thoroughly wash the slide to get rid of any remaining fixative.
The slide should be dehydrated by applying increasing alcohol concentrations (70%, 90%, and 100%) for five minutes at a time.
Let the slide air dry entirely.
Protocol for In-Situ Hybridization (ISH)
Setting Up the Probe
Create and assemble a particular probe that is complimentary to the desired target sequence.
Use a detectable signal to mark the probe, such as fluorescent dye, digoxigenin (DIG), or biotin.
To enable appropriate hybridization to the target sequence, denature the probe by heating it to 95°C for five minutes and then slowly chilling it.
The Pre-Hybrid Phase:
Pre-hybridize the slide using a blocking solution (e.g., 50% formamide, 2x SSC, 10% dextran sulfate, 1 mg/mL tRNA) at 37°C for at least 30 minutes in order to prevent non-specific binding sites on the chromosomes.
Rinse twice with SSC to remove the blocking solution gently.
The Use of Hybridization
Cover the pre-hybridized slide with a coverslip after applying the denatured probe to it.
To stop evaporation, seal the coverslip’s edges using rubber cement.
The slide should be left overnight in a humidified room at the proper hybridization temperature, which is typically 37°C or 42°C.
Washes After Hybridization
After carefully removing the coverslip, wash the slide twice at room temperature using SSC.
To remove the unbound probe, wash the slide for 15 minutes at 65°C with 0.1x SSC.
Indicator Recognition
For signal detection, employ the appropriate reagents based on the probe labeling technique.
Use anti-DIG antibody coupled to alkaline phosphatase or horseradish peroxidase for DIG-labeled probes.
Use streptavidin conjugated to either horseradish peroxidase or alkaline phosphatase for biotin-labeled probes.
Use the proper detection techniques for fluorescence probes according to the kind of fluorescent dye being utilized.
Visualization of Signals
When using alkaline phosphatase-conjugated antibodies, the signal may be seen as a colored precipitate by using a chromogenic substrate (such as NBT/BCIP).
When using horseradish peroxidase-conjugated antibodies, see the signal as a brown precipitate by using a chromogenic substrate (such as DAB).
Use a fluorescence microscope to see the signal for fluorescent probes.
Analysis and Microscopy
Using a light or fluorescence microscope, depending on the signal detecting technique used, examine the hybridized chromosomes.
Take pictures of the chromosomes and examine where the target sequence is located.
Utilize suitable software tools for processing and analyzing images in order to measure the distribution and intensity of the signal.
Troubleshooting
Ineffective Chromosome Dispersal:
Make certain that the larval stage is suitable for chromosomal preparation.
Maximize the duration and focus of the hypotonic therapy.
When squashing, use a smooth, clean slide.
Don’t apply too much pressure while squashing.
General Background Information
Modify the incubation period and composition of the blocking solution.
Achieve optimal temperatures and times for hybridization.
To remove an unattached probe, use the proper cleaning procedures.
Make sure the probe is labeled and produced correctly
Weak Indication
Boost the concentration of the probe.
Adjust the incubation period and signal detecting reagents as needed.
Make that the target sequence and the probe are complimentary.
When seeing signals with a microscope, use the right settings.
Polytene Chromosome ISH Applications
Gene mapping: Determine the precise chromosomal location of genes and regulatory elements using gene mapping.
Analyze gene expression: to see how genes are expressed throughout time and space.
Studies on genomic organization: Examine how genes are arranged on Polytene Chromosomes and other genomic characteristics.
Mutation analysis: Look for chromosomal deletions and rearrangements.
Evolutionary studies: Examine how genes are arranged and expressed differently in various species.
In summary
A thorough guidance for getting Drosophila polytene chromosomes ready for ISH is provided by this technique. Researchers may efficiently generate high-quality Polytene Chromosomes appropriate for examining gene expression, genomic structure, and other biological processes by closely adhering to each stage. Learning this method improves our comprehension of basic biological concepts and provides fascinating opportunities for examining the complex functions of the Drosophila genome.
Frequently Asked Questions
Write about Polytene Chromosomes?
Large chromosomes called polytenes are seen in the salivary glands of insects. Polytene Chromosomes were found by Balbiani in 1881. Polytene Chromosomes were found in the salivary glands, Polytene Chromosomes are often referred to as salivary gland chromosomes. Polytene Chromosomes are referred to as polytene because of their large number of chromonemata.
Define the Squashing?
Squashing is a method for preparing samples for viewing in microscopy. It entails pushing on a coverslip to flatten a sample of tissue or cell on a microscope slide.
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