nucleic acid extraction methods: dna - college of staten island

Molecular Diagnostics Fundamentals, Methods and Clinical ApplicationsSecond Edition

Copyright © 2012 F.A. Davis Company

Nucleic Acid Extraction Methods: DNA

Chapter 4



Objectives Compare and contrast organic, inorganic, and solid‐phase

approaches for DNA isolation. Compare and contrast organic and solid‐phase approaches for

isolating total RNA. Distinguish between the isolation of total RNA with that of

messenger RNA. Describe the gel‐based, spectrophotometric, and fluorometric

methods used to determine the quantity and quality of DNA and RNA preparations.

Calculate the concentration and yield of DNA and RNA from a given nucleic acid preparation.



Specimens Blood Buffy coat Bone marrow Solid tissue Lavage fluids Bacteria, viruses Fungi Organelles, mitochondria



Specimen Preparation Bone marrow, peripheral blood Density gradient centrifugation Differential osmolysis

Tissue Freeze/crush Mince Enzymatic digestion – proteinase K digestion

Plants/fungi Homogenize Vortex with glass beads



Isolation of DNA Preparing the Sample

Nucleated Cells in Suspension (Blood and Bone Marrow Aspirates) For differential density gradient centrifugation, whole blood or bone marrow

mixed with isotonic saline is overlaid with Ficoll. Upon centrifugation, the mononuclear WBCs (the desired cells for isolation of nucleic acid) settle into a layer in the Ficoll gradient that is below the less dense plasma components and above the polymorphonuclear cells and RBCs.

For differential lysis (differential osmotic fragility), Incubation of whole blood or bone marrow in hypotonic buffer or water will result in the lysis of the RBCs before the WBCs. The WBCs are then pelleted by centrifugation, leaving the empty RBC membranes (ghosts) and hemoglobin, respectively, in suspension and solution.



Isolation of DNA Preparing the Sample

Tissue Samples Grinding the frozen tissue in liquid nitrogen, homogenizing the tissue, or simply mincing the tissue using a

scalpel can disrupt whole tissue samples. Fixed, embedded tissue may be deparaffinized by soaking in xylene (a mixture of three isomers of

dimethylbenzene). Less toxic xylene substitutes, such as Histosolve, Anatech Pro‐Par, or ParaClear, are also often used for this purpose. After xylene treatment, the tissue is usually rehydrated by soaking it in decreasing concentrations of ethanol. Alternatively, fixed tissue may be used directly without dewaxing.

Tissue Fixatives Influencing Nucleic Acid QualityRelative Quality Average Fragment

Fixative of Nucleic Acid Size Range (kb) 10% buffered Good 2.0–5.0 neutral formalin Acetone Good 2.0–5.0 Zamboni’s Not as good 0.2–2.0 Clarke’s Not as good 0.8–1.0 Paraformaldehyde Not as good 0.2–5.0 Metharcan Not as good 0.7–1.5 Formalin‐alcohol‐ Not as good 1.0–4.0 acetic acid B‐5 less desirable <0.1 Carnoy’s less desirable 0.7–1.5 Zenker’s less desirable 0.7–1.5 Bouin’s less desirable <0.1



Isolation of DNA Preparing the Sample Microorganisms Enzymes lyzozyme or zymolyase ‐ digest cell wall polymers

Treatment with detergent (1 % sodium dodecyl sulfate) and strong base (0.2 M NaOH) in the presence of Tris base, ethylenediaminetetraacetic acid (EDTA), and glucose can break bacterial cell walls.

Boiling in dilute sucrose, Triton X‐1 00 detergent, Tris buffer, and EDTA after lysozyme treatment releasesDNA that can be immediately precipitated with alcohol

Mechanical force grinding or mixing with glass beads



Specimens Adequate for amplification by polymerase chain reaction (PCR)

Dried blood Saliva Bone, teeth Amniotic fluid Hair follicles, hair shafts Buccal cells Cerebrospinal fluid Fixed tissue Feces Soil



DNA Extraction Methods

Organic: uses organic chemicals, phenol, chloroform

Inorganic: uses inorganic chemicals, detergents, ethylenediamine tetraacetic acid (EDTA), acetic acid, salt (salting out, spooling)

Solid phase: DNA is immobilized on a solid support, beads, or columns



Organic DNA Isolation



Inorganic DNA Isolation



Solid‐Phase DNA Isolation Solid‐phase

isolation media include silica spin columns and beads.

Nucleic acid binding to silica beads is the basis for many automated extraction systems.



Other DNA Extraction Methods

Limiting specimens (fixed tissue, dried blood, bone) Rapid extraction for routine testing



Chelex removesmultivalent cations.

Heat tissue (hair roots, saliva, etc.) in 300 µL 5%–20% Chelex 100 resin [cation chelating resin].

DNA is insupernatant.

DNA Purification with Chelex Resin for PCR



DNA Extraction from Fixed Tissue for PCR



Mitochondrial DNA

Isolate mitochondria by centrifugation. Slow centrifugation Fast centrifugation

Isolate total DNA.



Methods to Assess DNA Gel electrophoresis with

known standards

Spectrophotometry1 OD260 = 50 μg/mL dsDNA(concentration)μg/mL x mL = μg DNA (yield)OD260 / OD280 ~ 1.6–2.0 (purity)

Genomic DNA should look like this:



Nucleic Acid Extraction Methods: RNA

Chapter 4



Laboratory Preparation Bench, equipment

Separate laboratory area designated “RNase Free” ( RNF). Wipe with RNase ZAP, RNase AWAY.

Disposables Certified RNase free Rinsed in 0.1% diethyl pyrocarbonate (DEPC)

Reagents Certified RNase free Add 0.05%–0.1% DEPC (except Tris) Test with RNase Alert (Ambion, Inc.)

Reactions Add RNasin (Promega)



Specimen Collection Bone marrow, peripheral blood Acid citrate dextrose (ACD) Liquid K3EDTA (Heparin) “RNA tubes”

Tissue Fresh in saline: process immediately Frozen, – 70°C, nitrogen Fixed, embedded



Specimen Preparation Bone marrow, peripheral blood Density gradient centrifugation Differential osmolysis (remove RBC or nuclei)

Tissue Freeze/grind Crush in denaturant

Bacteria/fungi/plants Homogenize Vortex with glass beads Enzymatic digestion (Zymolyase/lysozyme)



RNA Isolation

Organic Solid phase



Organic RNA Isolation GITC: strong RNAse denaturant



Solid‐Phase RNA Isolation



Macrodissect ormicrodissect

Digest with proteinase K

Organic extractionand precipitation

~20 micronsections

RNA Extraction from Fixed Tissue



Types of RNA Messenger RNA (mRNA) Ribosomal RNA (rRNA) Transfer RNA (tRNA) Heteronuclear RNA (hnRNA) Small nuclear RNA (snRNA) Double‐stranded RNA (dsRNA) Many small/micro RNAs



Messenger RNA (mRNA)

The efficiency of polyA and polyUbinding is variable.

Secondary structure in the target sample may compete with binding to the capture oligomer.

mRNAs with short polyA tails may not bind efficiently or at all.

AT‐rich DNA fragments might bind to the column and not only compete with the desired mRNA target but also contaminate the final eluant.

A A A A A A A A A

T T T T T T T T T

Bead or column



Methods to Assess RNA Gel electrophoresis (total RNA) (quality)

Spectrophotometry1 OD260 = 40 μg/mL RNA (concentration)μg/mL x mL= μg RNA (yield)OD260/ OD280 >1.6 (purity)

FluorometryRiboGreen

Total RNA should look like this.

--28S rRNA

--18S rRNA

M

M = molecular weight marker



Common Contaminants and Their Wavelengths of Peak Absorbance Wavelength (nm) Contaminant

230 Organic compounds 270 Phenol 280 Protein >330 Particulate matter



Fluorometry DNA‐specific dye Hoechst 33258 { 2‐[2‐(4‐hydroxyphenyl)‐(6‐benzimida‐zol)]‐6‐(1‐

methyl‐4‐piperazyl)‐benzimidazol/.3HCl} . This dye combines with adenine‐thymine base pairs in the minor groove of the DNA double helix and is thus specific for intact double‐stranded DNA.

PicoGreen and OliGreen (Molecular Probes, Inc.) are other DNA‐specific dyes that can be used for fluorometric quantification. Due to brighter fluorescence upon binding to double‐stranded DNA, PicoGreen is more sensitive than Hoechst dye.

OliGreen is designed to bind to short pieces of single‐stranded DNA (oligonucleotides). OliGreen will not fluoresce when bound to double‐stranded DNA or RNA.

Fluorometry measurements require calibration of the instrument with a known amount of standard before measurement of the sample.



Summary DNA is extracted by organic, inorganic, and solid‐phase

methods. DNA can also be extracted by more rapid methods or methods

designed for challenging specimens. RNA extraction methods include organic and solid‐phase

methods. mRNA can be specifically extracted using immobilized polyT or

polyU. DNA and RNA concentration, yield, and purity are assessed

using gel analysis, spectrophotometry, or fluorometry.

nucleic acid extraction methods: dna - college of staten island

Documents