ZOO: DSM-351 Practical
Unit 4: Molecular Biology
1. Study of Polytene Chromosomes
Polytene chromosomes are specialized giant chromosomes observed in the salivary glands of Dipteran larvae, such as Chironomus or Drosophila. These chromosomes are formed by repeated DNA replication without nuclear division (endoreduplication).
Objective
To prepare a temporary squash and identify the distinctive features of polytene chromosomes.
Identification Features
- Giant Size: Significantly larger than normal somatic chromosomes.
- Banding Patterns: Visible as alternating dark bands and light interbands.
- Chromocentre: The region where all the chromosomes are joined together.
- Puffs (Balbiani Rings): Swollen regions indicating active sites of transcription (RNA synthesis).
Procedure Summary
- Dissect the salivary glands from a healthy third-instar larva in physiological saline.
- Fix the tissue in Carnoy's fixative.
- Stain with Acetocarmine or Aceto-orcein.
- Prepare a squash mount and observe under high-power magnification.
2. Quantitative Estimation of DNA
Quantitative estimation determines the amount of DNA present in a given biological sample using optical density measurements.
Instrumentation
The estimation is performed using specialized optical instruments:
- Colorimeter: Used for colored chemical reactions like the Diphenylamine (DPA) test.
- Spectrophotometer: Measures the absorbance of UV light directly by the DNA molecules.
Methodology: Spectrophotometry
Principle: Nucleic acids (DNA/RNA) have a maximum absorbance of UV light at a wavelength of 260 nm due to the nitrogenous bases.
- A known volume of DNA solution is placed in a quartz cuvette.
- The absorbance (Optical Density) is recorded at 260 nm.
- Concentration is calculated based on the standard: 1.0 OD at 260 nm ≈ 50 µg/ml of double-stranded DNA.
Exam Focus: Practical Techniques and Calculations
Practical Tip: When using the colorimeter, always use a "reagent blank" (all chemicals except the DNA sample) to set the instrument to zero. This ensures the reading only accounts for the DNA concentration.
Common Error: Do not touch the transparent sides of the cuvette when using a spectrophotometer. Fingerprints can scatter light and lead to inaccurate (higher) absorbance readings.
Frequently Asked Questions (FAQs)
- Q: Why are polytene chromosomes called "giant" chromosomes?
A: Because they contain thousands of DNA strands bundled together through repeated replication cycles.
- Q: What is the significance of the 260/280 ratio?
A: It measures DNA purity. A ratio of ~1.8 indicates pure DNA; lower ratios suggest protein contamination.
- Q: Which stain is specific for DNA in chromosome studies?
A: Feulgen stain is often preferred as it is specific for DNA (the Feulgen reaction).