Question

Part of the process of purifying DNA from isolated chromosomes must involve: A. adding DNase, an enzyme that breaks apart DNA. B. adding proteases, enzymes that break apart proteins. C. adding RNase, an enzyme that breaks apart RNA. D. adding detergents, chemicals that break apart lipids.

Short answer

B. adding proteases, enzymes that break apart proteins.

Step-by-step solution

Understand the Goal of the Process

The aim of purifying DNA from isolated chromosomes is to remove contaminants and any other cellular components that are not DNA. This includes proteins, RNA, lipids, and other cellular debris.

Eliminate Incorrect Options

Options A, C, and D involve adding enzymes or chemicals that break apart molecules other than proteins. DNase breaks apart DNA, which is counterproductive in purifying DNA. RNase breaks apart RNA, and detergents break apart lipids, which could be relevant, but not the main source of contamination when purifying DNA from chromosomes.

Identify the Correct Option

Option B involves adding proteases, which are enzymes that break apart proteins. Proteins are the most likely contaminants when purifying DNA from chromosomes because chromosomal DNA is typically associated with histones and other DNA-binding proteins. Therefore, breaking apart these proteins is an essential step in the DNA purification process.

Key concepts

Chromosome Isolation

Chromosome isolation is a critical first step in DNA purification, a process designed to separate and extract chromosomal DNA from other cellular components. This includes separating DNA from the proteins, lipids, RNA, and various other molecules found in a cell.

To achieve this, scientists usually begin by lysing, or breaking open, the cells to release their contents. This is often done using a lysis buffer, which may contain specific chemicals that disrupt the cell membrane. However, care must be taken to preserve the integrity of the DNA during this process. After cell lysis, the mixture is typically subjected to a series of centrifugation steps. These are designed to fractionate the cellular components and allow the collection of a fraction enriched in chromosomes.

To ensure the success of chromosome isolation, it's essential to prevent the DNA from shearing and to minimize contamination by other cellular components. This lays a solid foundation for the subsequent steps of DNA purification.

Proteases

Proteases are a category of enzymes crucial in the process of DNA purification. They play a significant role in digesting proteins that may contaminate the DNA sample. In the context of chromosome isolation, proteases target histones and other DNA-binding proteins that are tightly associated with DNA.

Histones, which help package the DNA into nucleosomes, must be removed during DNA purification to obtain a clean sample of DNA. Adding proteases helps achieve this by breaking down these proteins into smaller peptides and amino acids. This enzymatic action ensures that the DNA can be separated from the protein contaminants, subsequently facilitating a purer DNA extraction.

Common Proteases in DNA purification

• Proteinase K: Widely used for its broad specificity and activity under a variety of conditions.
• Trypsin: Targets lysine and arginine residues and is often used in proteomics.

Proteases are added at a specific point in the protocol to ensure optimal activity and must be carefully controlled to avoid over-digestion, which could potentially harm the DNA.

Enzyme Application in DNA Extraction

The application of enzymes in DNA extraction is a technique utilized to remove unwanted components and isolate the target DNA from a sample efficiently. Enzymes such as proteases are used to degrade proteins, but there are others that specificity target different molecules.

For instance, RNase is employed to degrade RNA that may be present, ensuring it does not contaminate the DNA sample. However, in the process of purifying DNA, enzymes like DNase must be avoided as they would degrade the DNA itself, which is the very molecule we are aiming to purify. When enzymes are applied correctly, they enhance the purity and yield of the isolated DNA.

Key Considerations for Enzyme Use

• Enzyme purity and concentration are vital to control the reaction.
• The incubation times and temperatures must be optimized for each enzyme.
• Inhibitors may be required post-reaction to halt enzyme activity.

By understanding and controlling enzyme application, researchers can isolate high-quality DNA suitable for use in various downstream applications such as cloning, sequencing, and PCR.