ZOO: DSC-354 Practical - IV
Unit 1: Molecular Biology
1. Study of Polytene Chromosomes
Polytene chromosomes are giant chromosomes commonly found in the salivary glands of Dipteran insects like Chironomus or Drosophila larvae. They are excellent models for studying gene expression and chromosomal structure.
Objective
To identify and study the structure of giant polytene chromosomes from larval tissues.
Key Characteristics
- Endoreduplication: These chromosomes are formed by repeated rounds of DNA replication without cell division.
- Banding Pattern: They show distinct dark bands (chromomeres) and light interbands.
- Chromosome Puffs: Large swellings called "puffs" (or Balbiani rings) represent sites of active RNA transcription.
Practical Procedure (Squash Preparation)
- Select a healthy third-instar larva of Chironomus or Drosophila.
- Dissect the salivary glands in a drop of physiological saline.
- Fix the glands in 45% acetic acid or Carnoy's fixative.
- Stain with Acetocarmine or Aceto-orcein.
- Apply a coverslip and perform a gentle "squash" to spread the chromosomes.
- Observe under a compound microscope to identify the characteristic banding and puffs.
2. Quantitative Estimation of DNA
This procedure is used to determine the concentration of DNA in a biological sample using optical methods.
Methods of Estimation
The syllabus highlights two primary instruments for this purpose:
- Colorimeter: Measures the intensity of color produced by a chemical reaction (e.g., Diphenylamine reaction).
- Spectrophotometer: Measures the absorbance of light at specific wavelengths (typically 260 nm for DNA).
The Diphenylamine (DPA) Method
This is a common colorimetric assay for DNA estimation.
- Principle: When DNA is heated with acid, the deoxyribose sugar is converted to hydroxylevulinic aldehyde, which reacts with Diphenylamine to produce a blue-colored complex.
- Calculation: The intensity of the blue color is proportional to the DNA concentration, which is determined by comparing the absorbance of the unknown sample against a standard curve of known DNA concentrations.
Exam Focus: Practical Tips and FAQs
Exam Tip: In your practical record, ensure you draw the Chironomus polytene chromosomes with clearly labeled bands, interbands, and Balbiani rings. Examiners frequently ask about the significance of "puffs."
Common Pitfall: When using a spectrophotometer, remember that DNA absorbs maximally at 260 nm. Contamination by proteins is checked at 280 nm. A pure DNA sample has an A260/A280 ratio of approximately 1.8.
Frequently Asked Questions (FAQs)
- Q: Why are polytene chromosomes found in salivary glands?
A: The massive size allows for the high levels of protein production (saliva/glue) needed by the larvae during development.
- Q: What is the purpose of "squashing" the tissue?
A: To spread the overlapping chromosomes into a single layer for clear microscopic visualization.
- Q: What does a high absorbance at 260 nm indicate?
A: It indicates a high concentration of nucleic acids in the sample.