sample preserving adv lab tech

What is the key structural difference between DNA and RNA that makes RNA more susceptible to degradation?

RNA has a hydroxyl group (-OH) on the 2' carbon of its ribose sugar. This makes it highly susceptible to nucleophilic attack and is the dominant site for degradation.

Why is single-stranded RNA (ssRNA) the easiest form of nucleic acid to degrade?

Because it is single-stranded, fully exposed, and flexible, with its vulnerable 2'-OH group accessible along the entire backbone.

Why is double-stranded DNA (dsDNA) harder to degrade than ssRNA?

The double helix provides a protective, more rigid structure that shields the sensitive sugar-phosphate backbone from attack.

How does the structure of dsRNA differ from dsDNA, and what is the consequence?

In dsRNA, the 2'-OH group changes the backbone spacing and groove geometry. The major groove collapses and the minor groove opens, creating a less stable structure overall compared to dsDNA.

What is a major functional consequence of the major and minor grooves in DNA?

Most DNA-binding proteins read DNA sequences via the MAJOR groove.

How do the enzymes that degrade RNA (RNases) and DNA (DNases) differ in their stability?

RNase is more stable (heat-tolerant) than DNase, which can be inactivated by heat. This contributes to the higher risk of RNA degradation in samples.

What are the two main goals of sample preservation for nucleic acid isolation?

1) Limit the impact of degradation (from hydrolysis, pH, enzymes). 2) Prevent contamination (from bacteria/fungi).

What is the first principle of preserving nucleic acids?

Freeze the sample (the colder the better).

What is the second principle of preserving nucleic acids?

Use chemical preservatives like Ethanol, DMSO, or RNAlater.

Why must preservation choice be carefully considered?

Because the chemicals used for preservation can interact with the reagents used during DNA/RNA extraction, and can influence the choice and success of the extraction protocol.

According to the cited study (Flournoy et al. 1996), which alcohol is most efficient at denaturing DNases?

Ethanol > Methanol (Ethanol is faster/more efficient).

What is the order of penetration ability for primary alcohols into cell membranes?

Isopropanol → Ethanol → Methanol (Isopropanol penetrates best, Methanol penetrates slowest).

What is the general mechanism by which alcohols (like ethanol) preserve samples?

Alcohols dehydrate samples. Water (H₂O) moves out of the cells, which limits water movement and enzyme activity within the cell, halting degradation.