Methods for extracting Nucleic acids

Flashcards covering key concepts from the DNA extraction lecture notes, including sample sources, ancient DNA, DNA quantification, extraction methods, buffers, enzymes, PCI, silica-based methods, Chelex, and RNA extraction.

What are the five main DNA extraction method categories discussed in the notes?

Intro; Tissue homogenization and cell lysis; Organic (PCI) extraction; Solid-phase silica-based methods (e.g., QIAGEN spin columns); Chelex extraction.

What are common sample sources from which DNA may be isolated?

Blood, tissue, semen, saliva, urine, hair (root & shaft), teeth, bone, cigarette butts, envelopes/stamps, fingernail clippings, chewing gum, bite marks, feces; epithelial cells lining cheeks are a common human DNA source.

Why is ancient DNA often considered challenging/inauthentic?

Ancient DNA degrades by fragmentation, cross-linking, and deamination; postmortem mutation rate increases with time; some reported ancient-DNA sources (e.g., dinosaurs) are fiction.

What is the oldest authentic ancient DNA sequenced to date?

Approximately 2.5 million-year-old environmental DNA from Greenland sediments.

How much DNA does a diploid human cell contain?

Approximately 6 picograms (pg).

How much DNA is in a sperm cell?

Approximately 3 pg.

What is the theoretical DNA recovery per liter of blood?

30–60 ng.

What is the typical DNA yield range from standard isolation kits per sample?

About 3 µg to 260 µg of total genomic DNA.

How much DNA is typically required per standard PCR reaction?

Approximately 1 ng.

What does 1 ng of DNA roughly correspond to in a blood sample and in sperm?

Mass of DNA in 1/20 of 1 µL of blood or about 350 sperm.

What amount of DNA do Next-Generation Sequencing library prep protocols often specify per sample?

Approximately 20–500 ng of high-molecular-weight genomic DNA.

Name the five essential components common to all DNA extraction methods.

1) Tissue homogenization; 2) Cell lysis; 3) Remove or inhibit nucleases; 4) Separate & remove other biological macromolecules and inhibitors; 5) Precipitate & resuspend/elute DNA.

What are two common tissue disruption methods?

Bead beating with grinding media; manual grinding with a micropestle.

What does a typical lysis buffer contain?

A strong detergent (e.g., SDS or CTAB) plus buffering salts (e.g., Tris-HCl) and ionic salts (e.g., NaCl).

Which detergent is commonly used in many lysis buffers?

SDS (sodium dodecyl sulfate).

What is the role of EDTA in lysis buffers?

Chelates divalent cations like Mg2+, inactivating DNases.

Why is Proteinase K added during lysis?

To digest proteins, including DNases and RNases.

What temperatures are typical for Proteinase K digestion?

Approximately 37–65°C for 30 minutes to 24 hours.

After Proteinase K digestion, what is typically done?

Centrifuge to pellet debris; transfer the supernatant to a new tube.

Which extraction method is introduced after PK digestion in the notes?

Phenol/Chloroform/Isoamyl Alcohol (PCI) liquid-liquid extraction.

What are the three phases in PCI extraction?

Aqueous phase (DNA/RNA), Interphase (proteins), Organic phase (lipids and some proteins).

What reagents make up PCI?

Phenol, chloroform, isoamyl alcohol.

What is the typical Phenol:Chloroform:Isoamyl Alcohol ratio in PCI?

25:24:1.

Why is a second extraction with 24:1 chloroform:isoamyl alcohol used after PCI?

To remove traces of phenol.

What is the purpose of ethanol precipitation in PCI extraction?

Concentrate DNA by precipitating it with ethanol in the presence of salt.

What salts are typically used for DNA precipitation in PCI extraction?

Sodium acetate (or other monovalent salts).

What is the purpose of the 70% ethanol wash during DNA precipitation?

To remove solutes and impurities from the DNA pellet.

How should the ethanol wash be performed to avoid losing DNA?

Add 70% ethanol until the tube is about 2/3 full; vortex briefly and re-centrifuge.

What buffer is used to resuspend DNA after PCI extraction?

TE buffer or TE-4 (Tris-EDTA buffers).

What is the purpose of EDTA in DNA storage buffers?

Chelates Mg2+ to help inactivate DNases and protect DNA.

What are the pros of PCI extraction?

Yields potentially large amounts of relatively pure, high-molecular-weight DNA; double-stranded; relatively inexpensive.

What are the cons of PCI extraction?

Time-consuming; multiple tube transfers increase contamination risk; uses hazardous chemicals.

How does solid-phase silica-based DNA extraction work in QIAGEN spin columns?

Lysate is combined with ethanol and high salt; DNA binds to silica membrane under high ionic strength; contaminants pass through; DNA is washed and eluted.

What are the pros of silica-based DNA extraction?

Moderately fast; safe; yields highly purified double-stranded DNA.

What are the cons of silica-based DNA extraction?

Requires multiple tube transfers; kits can be expensive; chemistry is often proprietary and may limit visibility; yields may be insufficient for some NGS applications.

What is Chelex 100 and its role in DNA extraction?

An ion-exchange resin (styrene-divinylbenzene copolymers with iminodiacetate groups) that chelates Mg2+ to inactivate nucleases and protect DNA.

What are the basic steps of Chelex extraction?

Add 5% Chelex; incubate at 56°C for 30 minutes; heat to 100°C for 8 minutes; centrifuge and collect the supernatant (DNA in the supernatant).

What type of DNA does Chelex extraction yield?

Single-stranded DNA.

What are the pros and cons of Chelex extraction?

Pros: quick/easy; minimal homogenization; single-tube; reduces contamination. Cons: yields single-stranded DNA; not ideal for many downstream applications; residual Chelex can inhibit PCR; relatively expensive as a trademarked kit.

What is the most common RNA extraction method?

Guanidinium thiocyanate-phenol-chloroform extraction.

Why is guanidinium thiocyanate used in RNA extraction?

It denatures/eliminates RNases, protecting RNA.

What is required to create a cDNA library from RNA?

Reverse transcriptase to synthesize complementary DNA (cDNA) from RNA.

Why must RNA samples be fresh or rapidly frozen?

RNases rapidly degrade RNA; tissues can lose integrity within about an hour after death if not preserved.