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Copyright 2010 American Society of Cytopathology

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American Society of Cytopathology Core Curriculum in Molecular Biology


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American Society of Cytopathology Core Curriculum in Molecular Biology

Chapter 3

Molecular TechniquesNucleic Acid Extraction Methods

Keisha N. Brooks, MS, CT, MB(ASCP)University of Tennessee Health Science CenterMemphis, Tennessee

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DNA/RNA ExtractionPurpose: to release the nucleic acid

from the cell for use in subsequent

procedures.

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Specimens

• Blood (20–60µg DNA/ml)• Buffy coat (100–250µg/ml)• Bone marrow• Solid tissue (2–25µg DNA/mg)• Lavage fluids• Bacteria, viruses• Fungi• CSF (Cerebral spinal fluid)

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Specimens (cont.)

– Dried blood (75–150ng DNA/spot)– Saliva (5–15µg DNA/ml)– Bone, teeth– Amniotic fluid, (>8 weeks)– Hair follicles, hair shafts – Buccal cells (2µg DNA/mg)– Cerebrospinal fluid– Fixed tissue– Feces– Soil

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Prenatal testing• Amniotic fluid‐prenatal determination of genetic disorders; 15‐20 weeks gestation

• Chorionic villus sampling‐ 10‐12 weeks gestation• Pre‐implantation testing‐test embyros for a genetic disease before they are implanted

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Pre-Treatment of Nucleic AcidsInitial steps depend on:1. Starting material 2. Target Examples: viscous samples such as sputum;

bacteria such as strep pneumoniae have a thick polysaccharide capsule

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Basic Steps in Isolating DNA from Clinical Specimens

• Separate WBCs from RBCs, if necessary

• Lyse WBCs or other nucleated cells

• Denature/digest proteins

• Separate contaminants (e.g., proteins)from DNA

• Precipitate DNA (alcohol)

• Re‐suspend DNA in final buffer

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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

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Liquid Phase Organic Extraction• Cell lysis of sample: detergent (example: sodium dodecyl

sulfate)• Lysed samples mixed with phenol/chloroform isoamyl alcohol

mixture; two layers are formed.• Proteins and lipids settle to bottom layer.• DNA is removed with top aqueous layer. • DNA is precipitated with alcohol and rehydrated.• Disadvantages: slow, labor‐intensive, toxic (phenol,

chloroform), fume hood required, disposal of hazardous materials required

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Liquid Phase Organic Extraction

Lysis(SDS)

Hydrophilic layer (top: DNA)Hydrophobic layer (bottom: lipids, proteins)

DNA precipitation(ethanol or isoproanol,high concentration of salt)

Phenol, chloroform, centrifuge

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Liquid Phase Inorganic Extraction

• Cell membranes are lysed using SDS• RNA is removed with RNase. • Proteins are precipitated with salt solution.• DNA is precipitated with alcohol and rehydrated.

• Advantages: fast and easy method, uses nontoxic materials, no fume hood required, no hazardous materials disposal issues, produces high‐quality DNA

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Liquid Phase Inorganic Extraction

Lysis(Tris, EDTA,SDS) Protein

precipitation(sodiumacetate)

DNAprecipitation(isopropanol)

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Solid-phase extraction

• Cell membrane lysis• Protein digestion (example: proteinase‐K)• DNA is precipitated by addition of alcohol• Passed thru silica –impregnated filter‐binds

and purifies DNA • Washing steps• DNA eluted in nuclease free water

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Which method?

• Specimen type and its volume– What is the size or volume of each sample?– Amount of DNA or RNA required– Equipment and tube sizes required

• Processing speed• Required yield of DNA/RNA• Required purity of DNA/RNA• Ease of operation• Cost of preparation• Shelf life/storage conditions• Hazardous reagents?

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DNA Extraction from Fixed Tissue

~20 micronsections

Deparaffinize(xylene, ethanolwash)

Digest(proteinase K,Tris buffer)

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RNA• RNA analysis is required for studies on gene expression and some viruses such as HIV

• RNA –less stable than DNA – It is easily degraded by RNase enzymes.

– Use sterile, disposable plasticware (tubes, filter tips) marked “For RNA Use Only”.

– Always wear gloves and work in a hood whenever possible/practical.

• Extraction methods are similar to DNA – organic and solid phase methods

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Basic Steps in IsolatingRNA from Clinical Specimens

Separate WBCs from RBCs, if necessary

Lyse WBCs or other nucleated cells

Denature/digest proteins

Separate proteins, DNA, and contaminantsfrom RNA

Precipitate RNA (alcohol)

Re‐suspend RNA in final buffer

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Possible problems: RNA Extraction

• Cross contamination of specimens• RNA‐ easy to change due to presence of RNases – proteins can renature after heating and become active again

• Inconsistent results

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Nucleic Acid Analysis

DNA or RNA is characterized using several different methods for assessing quantity, quality, and

molecular size.

• UV spectrophotometry

• Agarose gel electrophoresis

• Fluorometry

• Colorimetric blotting

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Components of a Spectrophotometer

Stable source of radiant energy– UV, hydrogen lamp– Visible, tungsten filament lamp

• System of lenses and mirrors to focus the beam• Monochromator (resolves radiation into wavelength bands)

• Transparent container for sample• Detector• Meter or recorder

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Spectrophotometer

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Concentration of Nucleic Acids

• DNA and RNA absorb maximally at 260 nm.• Proteins absorb at 280 nm.• Using the Beer‐Lambert Law, nucleic acid concentration can be determined from the absorptivity constants for DNA and RNA– Absorptivity constant is 50 for DNA; 40 for RNA.

• Spec reading X 50 x dil factor= µg/ml of DNA

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Example: DNA Concentration

• A DNA preparation diluted 1:100 yields an absorbance reading of 0.200 at 260 nm. What is the DNA concentration?

• Concentration = 0.200 absorbance units x 50 µg/ml per absorbance x 100 = 1000 µg/ml

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Multiply the concentration of theDNA or RNA sample by the

volume of hydrating solution added.

Example for DNA: 150 µg/mL X 0.1 mL = 15 µg

Concentration from UV Spec. (µg DNA per ml of hydrating

solution)

Volume of hydration solution

DNA yield

Quantity from UV SpectrophotometryCalculating Yield

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Quality (Purity) of DNA• Nucleic acids absorb light at 260.• Protein absorbs light at 280 nm.• 260:280 ratio is the purity of the nucleic acid.• Ratio should be 1.6‐2.0.• If value is less than 1.6, possible protein contamination; repeat protein removal step of isolation procedure

• If value greater than 2.0, RNA contamination. Use RNase to remove contamination

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Quality (Purity) of RNA

• RNA tolerates a higher 260:280 nm ratio– Acceptable range: 2.0‐2.3– < 1.7 = protein or other contamination– Difficult to detect DNA contamination in RNA preparations

• Treat with DNase to avoid DNA contamination

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Example: DNA Quality

• A DNA preparation has the following A260 and A 280 reading:

• A 260 = 0.419 A 280 = 0.230• Is this preparation suitable for further use?

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Answer:

• A 260/ A 280 = 0.419 / 0.230 = 1.82 ‐ this is a suitable sample, because the value is between 1.6 and 2.0

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Troubleshooting Nucleic Acid Preparation Methods

• Problem: No or low nucleic acid yield.– Make sure that ample time was allowed for re‐suspension or

rehydration of sample.or

– Repeat isolation from any remaining original sample (adjust procedure for possible low cell number or poorly handled starting material).

or– Concentrate dilute nucleic acid using ethanol precipitation.

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Troubleshooting (cont.)

• Problem: Poor nucleic acid quality– If sample is degraded, repeat isolation from remaining

original sample, if possible.or

– If sample is contaminated with proteins or other substances, clean it up by re‐isolating (improvement depends on the extraction procedure used).

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Summary

• DNA and RNA are 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.

• DNA and RNA concentration, yield, and purity can be assessed by spectrophotometry.

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