Sample Preparation & Nucleic Acid Extraction Flashcards

What is the goal of nucleic acid extraction?

Answer: To isolate DNA or RNA from samples

The goal of nucleic acid extraction is to isolate and purify DNA or RNA from biological samples. This process separates the nucleic acids from other cellular components like proteins, lipids, and carbohydrates, which can interfere with downstream molecular applications. Obtaining a clean, high-quality sample is essential for accurate results in techniques such as PCR, sequencing, or cloning.

Why must samples be free of contaminants before PCR?

Answer: They can inhibit enzyme activity

Samples must be free of contaminants before PCR because various substances can inhibit enzyme activity. Contaminants like proteins, salts, or other cellular debris can interfere with the Taq polymerase, preventing efficient DNA amplification. Ensuring a clean sample is crucial for obtaining reliable, specific, and accurate PCR results, avoiding false negatives or reduced yield.

Which chemical helps lyse cells during DNA extraction?

Answer: Detergent (SDS)

Detergents, such as SDS (Sodium Dodecyl Sulfate), are commonly used in DNA extraction to help lyse cells. They disrupt the lipid bilayers of cell membranes and nuclear envelopes, effectively breaking open the cells and releasing their contents, including DNA. This initial lysis step is critical for making the DNA accessible for subsequent purification steps.

What is the purpose of using proteinase K in DNA extraction?

Answer: Degrades proteins in the sample

Proteinase K is an enzyme used in DNA extraction to degrade proteins present in the sample. Its primary purpose is to break down cellular proteins, including nucleases that could degrade DNA, and histones that are tightly bound to DNA. This enzymatic digestion helps to purify the DNA by removing protein contaminants, ensuring a cleaner and more stable DNA sample.

What role does ethanol play in DNA extraction?

Answer: Precipitates and isolates DNA

Ethanol plays a crucial role in DNA extraction by precipitating and isolating DNA from the aqueous solution. When added to the DNA solution, it reduces the solubility of DNA, causing it to clump together and become visible. This allows the DNA to be easily pelleted by centrifugation, separating it from soluble contaminants and facilitating its purification.

Why is centrifugation used in DNA extraction?

Answer: To pellet and separate DNA

Centrifugation is used in DNA extraction primarily to pellet and separate DNA from the solution. After DNA has been precipitated (e.g., with ethanol), centrifugation applies centrifugal force to compact the DNA into a solid pellet at the bottom of the tube. This allows for the easy removal of the supernatant containing unwanted contaminants, effectively purifying the DNA.

Which sample type is commonly used for PCR?

Answer: Blood or tissue

Blood and tissue are commonly used sample types for PCR because they contain cells rich in DNA. PCR can amplify specific DNA sequences from these biological materials, making it invaluable for applications like forensic analysis, genetic disease diagnosis, and pathogen detection. The quality and quantity of DNA extracted from these samples are critical for successful PCR amplification.

What is the role of a lysis buffer?

Answer: Disrupts membranes and protects nucleic acids

A lysis buffer's role is to disrupt cell membranes and protect nucleic acids during extraction. It typically contains detergents to break open cells and release their contents, along with other components like chelating agents or proteases. These components help to inactivate enzymes that could degrade DNA or RNA, ensuring the integrity of the nucleic acids for downstream applications.

How is RNA contamination prevented in DNA extraction?

Answer: Add RNAse enzyme

RNA contamination in DNA samples can interfere with DNA-based applications like PCR. To prevent this, RNAse (ribonuclease) enzyme is commonly added during DNA extraction protocols. RNAse specifically degrades RNA molecules into smaller fragments, ensuring that the final purified sample contains only DNA, which is crucial for accurate and specific DNA analysis.