Nucleic Acid Isolation – Key Concepts

These flashcards review essential concepts, reagents, sample types, and quality-control methods involved in the isolation and analysis of DNA and RNA, as discussed in the lecture notes.

Where in the cell is DNA primarily located?

In the nucleus.

Where in the cell is RNA mostly located?

In the cytoplasm.

What is the main goal of nucleic acid extraction?

To obtain purified nucleic acid free of proteins, carbohydrates, lipids, and other contaminants.

What are the three major steps in nucleic acid isolation?

(1) Cell lysis, (2) purification/separation of the nucleic acid, (3) verification of quality and quantity.

Who first isolated DNA from human cells and in what year?

Friedrich Miescher, 1869.

Which scientists demonstrated semi-conservative DNA replication and when?

Meselson and Stahl, 1958.

For what plasmid size range was alkaline lysis originally designed?

1–50 kb plasmid DNA.

Why are Gram-positive bacteria harder to lyse than Gram-negative bacteria?

They possess a thick peptidoglycan cell wall that resists chemical lysis.

Which blood cells are targeted when extracting DNA from whole blood?

White blood cells (leukocytes).

Name two non-blood human samples commonly used for DNA testing.

Hair follicles and cheek (buccal) swabs (bones/teeth can also be used).

What main obstacle must be overcome when extracting DNA from bacteria and fungi?

Breaking their tough cell walls to release nucleic acid.

What is the function of Proteinase K in DNA extraction?

It digests proteins, the chief contaminants in DNA preparations.

Which enzyme specifically lyses bacterial cell walls during DNA extraction?

Lysozyme.

What detergent is used at 1 % in alkaline extraction to reduce surface tension?

Sodium dodecyl sulfate (SDS).

List the four key reagents in alkaline extraction of DNA.

1 % SDS, 0.2 M NaOH, EDTA, and glucose.

Give two mechanical disruption methods used for tough specimens.

Grinding with mortar and pestle, or agitation with glass beads.

Name the main components of the boiling extraction buffer used with lysozyme.

Diluted sucrose, Triton X-100, Tris buffer, and EDTA.

Why are plasma specimens useful for viral nucleic acid detection?

Cell-free viruses and viral nucleic acids circulate in plasma, simplifying extraction.

What are exosomes and which nucleic acid do they often carry for testing?

Small vesicles released by cells that frequently contain mRNA.

Which reagent is used in density-gradient centrifugation to isolate white cells?

Ficoll, a highly branched sucrose polymer.

In hypotonic solutions, which blood cells lyse first—RBCs or WBCs?

Red blood cells lyse first; prolonged exposure also lyses WBCs.

Which solvent removes paraffin from fixed tissue blocks before DNA extraction?

Xylene, followed by graded ethanol rehydration.

Name the three principal DNA isolation chemistries.

Organic, inorganic ("salting out"), and solid-phase isolation.

What two organic solvents are mixed in equal parts for traditional organic DNA extraction?

Phenol and chloroform.

Which quaternary ammonium compound (abbrev. CTAB) helps degrade fungal cell walls?

Cetyltrimethylammonium bromide.

Why is RNase a troublesome lab contaminant and how is it controlled?

RNases are ubiquitous and stable; they are inhibited by guanidinium thiocyanate or other RNase inhibitors.

During organic extraction, which layer (aqueous or organic) contains DNA?

The upper aqueous layer.

Why is 70 % ethanol used after absolute ethanol precipitation of DNA?

To wash away residual salts while keeping DNA insoluble.

What is meant by "salting out" in DNA isolation?

An inorganic method that precipitates proteins with high salt at low pH instead of using organic solvents.

What material in solid-phase isolation binds DNA under high-salt conditions?

Silica (e.g., in diatomaceous earth, columns, or beads).

List the five basic steps in silica-column solid-phase DNA extraction.

Lysis, addition of high-salt/ethanol, adsorption to silica, washing, and elution.

For what purpose is Chelex resin commonly used?

Rapid DNA extraction from minimal forensic samples by boiling with a 10 % Chelex suspension.

How is mitochondrial DNA separated from other organelles before lysis?

Differential centrifugation: low-speed spin removes debris, high-speed spin pellets mitochondria.

Why is RNA isolation more challenging than DNA isolation?

RNA is readily degraded by RNases that easily renature and remain active.

Name the chemical most often included as an RNase inhibitor in lysis buffers.

Guanidinium thiocyanate (GITC).

What is the standard phenol:chloroform:isoamyl alcohol ratio for RNA extraction?

25 : 24 : 1.

At what pH should the organic phase be kept during RNA extraction to minimize DNA carryover?

Acidic, pH 4–6.

Which structural feature of mRNA enables its selective capture on polyT columns?

The 3′ poly-A tail that hybridizes to poly-T (or poly-U) oligomers.

Roughly how much mRNA is obtained from 1 µg of total RNA?

About 30–40 ng.

Which fluorescent dye specifically stains double-stranded DNA in gels?

SybrGreen I (ethidium bromide also stains but is not dsDNA-specific).

What two rRNA bands indicate intact RNA on an agarose gel?

28S rRNA (large) and 18S rRNA (small) bands.

At what wavelength do nucleic acids maximally absorb light for spectrophotometry?

260 nm.

What 260/280 absorbance ratio signifies relatively pure nucleic acid?

A ratio of ≥ 1.8.

Which fluorescent dye is specific for intact double-stranded DNA and detects as little as 200 ng/mL?

Hoechst 33258.

What technology uses microchannels on a chip to size and quantify nucleic acids?

Microfluidics “lab-on-a-chip” systems.