Untitled Deck Flashcards
(26 cards)
Why is studying DNA essential for understanding and treating human diseases?
Studying DNA allows researchers to identify genetic factors underlying diseases, enabling targeted treatments and personalized medicine.
How does the purity and quantity of extracted DNA affect clinical research?
Purity and quantity of DNA are crucial for accurate downstream applications like PCR, as contaminants can inhibit reactions and compromise results.
List four different types of samples from which human DNA can be isolated.
Blood, buccal cells, hair follicles, and tissue biopsies.
Why are buccal cells considered a convenient source for DNA extraction?
Buccal cells are non-invasive to collect and provide sufficient DNA for analysis.
How does PBS preserve cells during DNA extraction?
PBS prevents cell degradation by maintaining an isotonic environment.
Name the three common methods of DNA extraction.
Phenol-chloroform, silica-based, and magnetic bead methods.
What are the advantages of silica-based extraction over other methods?
Silica-based methods are faster, avoid toxic chemicals, and yield high-quality DNA.
Why is it important to ensure no food or drink is consumed 30 minutes before buccal swab collection?
To prevent contamination of the DNA sample with food particles or foreign DNA.
What is the purpose of adding proteinase K and lysis buffer during the lysis step?
Proteinase K breaks down proteins, while lysis buffer disrupts cell membranes and denatures proteins, releasing DNA.
How does ethanol enhance DNA binding to the silica membrane?
Ethanol removes water, creating conditions that favor DNA binding to silica.
Why is it important to incubate the sample at 56°C during lysis?
Incubation at 56°C activates proteinase K and ensures efficient cell lysis.
During the washing step, why are two different buffers used?
To sequentially remove proteins, salts, and other impurities.
What is the role of the elution buffer in the final step of DNA extraction?
The elution buffer releases bound DNA from the silica membrane for collection.
What is the role of proteinase K in DNA extraction?
Proteinase K digests proteins, including histones, freeing DNA from protein complexes.
How do chaotropic salts in the lysis buffer aid in DNA isolation?
Chaotropic salts disrupt hydrogen bonds and solubilize cellular components, aiding DNA release.
Why is absolute ethanol necessary during the DNA binding step?
Absolute ethanol promotes DNA precipitation and binding to the silica membrane.
What does the pre-wash solution remove from the sample?
The pre-wash solution removes residual salts and contaminants.
What is the purpose of the wash solution?
To clean the DNA-bound silica by removing remaining impurities.
How does the elution buffer facilitate DNA retrieval from the silica membrane?
The elution buffer rehydrates the DNA, releasing it from the silica surface.
Why is it important to determine the concentration and purity of extracted DNA before using it in experiments?
Ensuring proper concentration and purity prevents reaction failures and ensures reliable experimental results.
How does the A260/A280 ratio indicate DNA purity?
The A260/A280 ratio measures protein contamination, with pure DNA having a ratio of ~1.8.
What are the acceptable values for the A260/A280 ratio, and what do they signify?
Ratios of 1.8–2.0 indicate pure DNA, while lower values suggest protein contamination.
Why is it necessary to blank the spectrophotometer with a blanking solution before measuring DNA concentration?
To account for background absorbance and ensure accurate readings.
What does an A260/A280 ratio below 1.7 indicate about your DNA sample?
The sample is contaminated with proteins.
