Unit 6 Nucleic Acid Extraction Methods

Unit 6 Nucleic Acid Unit 6 Nucleic Acid Extraction MethodsExtraction Methods

Terry Kotrla, MS, MT(ASCP)BBTerry Kotrla, MS, MT(ASCP)BBFall 2007Fall 2007

PurposePurpose►To release nucleic acid from the cell To release nucleic acid from the cell

for use in other proceduresfor use in other procedures►Must be free from contamination with Must be free from contamination with

protein, carbohydrate, lipids or other protein, carbohydrate, lipids or other nucleic acids.nucleic acids.

►Used pure nucleic acids for testing.Used pure nucleic acids for testing.

IsolationIsolation►Routinely isolated from human, fungal, Routinely isolated from human, fungal,

bacterial and viral sources.bacterial and viral sources.►Pretreat to make nucleated cells Pretreat to make nucleated cells

available,available, whole bloodwhole blood Tissue samplesTissue samples MicroorganismsMicroorganisms

►Need sufficient sample for adequate Need sufficient sample for adequate yield.yield.

Organic IsolationOrganic Isolation►Must purify DNA by removing Must purify DNA by removing

contaminants.contaminants.►Accomplished by using combination of Accomplished by using combination of

high salt, low pH and an organic high salt, low pH and an organic mixture of phenol and chloroform.mixture of phenol and chloroform.

►To avoid RNA contamination add To avoid RNA contamination add RNAse, enzyme that degrades RNA.RNAse, enzyme that degrades RNA.

Phenol/ChloroformPhenol/Chloroform►Biphasic emulsion formsBiphasic emulsion forms►Hydrophobic layer on bottom has cell Hydrophobic layer on bottom has cell

debris.debris.►Hydrophilic layer on top has dissolved Hydrophilic layer on top has dissolved

DNADNA►Remove top layer, add cold ethanol, Remove top layer, add cold ethanol,

DNA precipitates out.DNA precipitates out.

Inorganic Isolation MethodsInorganic Isolation Methods► Also called “salting out”.Also called “salting out”.► Uses low pH and high salt condition to selectively Uses low pH and high salt condition to selectively

precipitate proteins.precipitate proteins.► DNA is left in solution (picture on far left).DNA is left in solution (picture on far left).► Precipitate out DNA with isoproproanol (middle Precipitate out DNA with isoproproanol (middle

and right side pictures).and right side pictures).► I know this is not scientific but the precipitated out I know this is not scientific but the precipitated out

DNA is usually referred to as “snotty”.DNA is usually referred to as “snotty”.

Solid Phase IsolationSolid Phase Isolation► More rapid and effectiveMore rapid and effective► Use solid matrix to bind the DNA.Use solid matrix to bind the DNA.► Wash away contaminants.Wash away contaminants.► Elute DNA from columnElute DNA from column► Textbook page 70, Figure 4.3Textbook page 70, Figure 4.3

Solid Phase IsolationSolid Phase Isolation► The diagram below explains the attractive The diagram below explains the attractive

properties of solid phase for DNA and RNA.properties of solid phase for DNA and RNA.

Crude LysisCrude Lysis►Used for:Used for:

Screening large numbers of samplesScreening large numbers of samples Isolation of DNA in limited amountsIsolation of DNA in limited amounts Isolation from challenging samples, ie, Isolation from challenging samples, ie,

paraffin embedded tissueparaffin embedded tissue►Usually not done in clinical laboratory.Usually not done in clinical laboratory.►Textbook page 71, Figure 4-4Textbook page 71, Figure 4-4

Isolation of Mitochondrial Isolation of Mitochondrial DNADNA

►MMitochondrial DNA is passed from itochondrial DNA is passed from generation to generation along the generation to generation along the maternal lineage. maternal lineage.

►Centrifugation to separate outCentrifugation to separate out►LyseLyse►Precipitate with cold ethanol.Precipitate with cold ethanol.

Isolation of RNAIsolation of RNA►Requires STRICT precautions to avoid Requires STRICT precautions to avoid

sample degradation.sample degradation.►RNA especially labile.RNA especially labile.

RNAsesRNAses►RNases are naturally occurring RNases are naturally occurring

enzymes that degrade RNAenzymes that degrade RNA►Common laboratory contaminant (from Common laboratory contaminant (from

bacterial and human sources)bacterial and human sources)►Also released from cellular Also released from cellular

compartments during isolation of RNA compartments during isolation of RNA from biological samples from biological samples

►Can be difficult to inactivateCan be difficult to inactivate

RNAsesRNAses►RNAses are enzymes which are small RNAses are enzymes which are small

proteins that can renature and proteins that can renature and become active.become active.

►MUST be eliminated or inactivated MUST be eliminated or inactivated BEFORE isolation.BEFORE isolation.

►CRITICAL to have a separate RNAse CRITICAL to have a separate RNAse free area of lab.free area of lab.

Protecting Against RNAseProtecting Against RNAse►Wear gloves at all timesWear gloves at all times►Use RNase-free tubes and pipet tipsUse RNase-free tubes and pipet tips►Use dedicated, RNase-free, chemicalsUse dedicated, RNase-free, chemicals►Pre-treat materials with extended heat Pre-treat materials with extended heat

(180 C for several hours), wash with (180 C for several hours), wash with DEPC-treated water, NaOH or H2O2DEPC-treated water, NaOH or H2O2

►Supplement reactions with RNase Supplement reactions with RNase inhibitorsinhibitors

Total RNATotal RNA►80-90% of total RNA is ribosomal RNA.80-90% of total RNA is ribosomal RNA.►2.5-5% is messenger RNA2.5-5% is messenger RNA

Organic RNA ExtractionOrganic RNA Extraction1.1. Lyse/homogenize cellsLyse/homogenize cells2.2. Add phenol:chloroform:isoamyl Add phenol:chloroform:isoamyl

alcohol to lysed sample, and alcohol to lysed sample, and centrifugecentrifuge

3.3. Organic phase separates from Organic phase separates from aqueous phaseaqueous phase Organic solvents on bottomOrganic solvents on bottom Aqueous phase on top Aqueous phase on top

(contains total RNA)(contains total RNA) Cellular debris and genomic Cellular debris and genomic

DNA appears as a “film” of DNA appears as a “film” of debris at the interface of the debris at the interface of the two solutionstwo solutions

4.4. Remove RNA solution to a Remove RNA solution to a clean tube; precipitate RNA clean tube; precipitate RNA and wash with ethanol, then and wash with ethanol, then resuspend RNA in waterresuspend RNA in water

Affinity Purification of RNAAffinity Purification of RNA1.1. Lyse cells, and spin to remove Lyse cells, and spin to remove

large particulates/cell debrislarge particulates/cell debris2.2. Apply lysate (containing nucleic Apply lysate (containing nucleic

acids and cellular contaminants) acids and cellular contaminants) to column with glass membraneto column with glass membrane

3.3. Wash with alcohol to remove Wash with alcohol to remove contaminants; nucleic acids contaminants; nucleic acids stick to glass membrane while stick to glass membrane while contaminants wash through. contaminants wash through. Treat with DNase enzyme to Treat with DNase enzyme to remove contaminating DNA.remove contaminating DNA.

4.4. Apply water to the column; Apply water to the column; purified RNA washes off the purified RNA washes off the glass and is collectedglass and is collected

Isolation of PolyA (messenger) Isolation of PolyA (messenger) RNARNA

►Only 2.5-5%Only 2.5-5%►mRNA molecules have a tail of A’s at mRNA molecules have a tail of A’s at

the 3’ end (polyA tail)the 3’ end (polyA tail)►Oligo(dT) probes can be used to purify Oligo(dT) probes can be used to purify

mRNA from other RNAsmRNA from other RNAs►mRNA can be eluted from oligo(dT) mRNA can be eluted from oligo(dT)

matrix using water or low-salt buffermatrix using water or low-salt buffer►Textbook page 75, Figure 4.7Textbook page 75, Figure 4.7

ElectrophoresisElectrophoresis►Analyze DNA and RNA for quality by Analyze DNA and RNA for quality by

electrophoresis.electrophoresis.►Fluorescent dyesFluorescent dyes

Ethidium bromide Ethidium bromide SybreGreenSybreGreen

►Appearance depends on type of DNA Appearance depends on type of DNA isolated.isolated.

►Will be covered in more detail in unit 7.Will be covered in more detail in unit 7.

Staining with Ethidium Bromide Staining with Ethidium Bromide and Sybr Green and Sybr Green

► DNA is electrophoresed and stained. DNA is electrophoresed and stained. ► Left – Ethidium Bromide Right – Sybr GreenLeft – Ethidium Bromide Right – Sybr Green► The lane in the left side of the picture on the left is The lane in the left side of the picture on the left is

a “ladder” which has fragments of a “ladder” which has fragments of knownknown base pair base pair (bp) sizes. This allows determination of the size of (bp) sizes. This allows determination of the size of isolated fragments.isolated fragments.

SpectrophotometrySpectrophotometry► Sample absorbances are determined on the Sample absorbances are determined on the

spectrophotometer at 260nm and 280nmspectrophotometer at 260nm and 280nm nucleic acid (DNA, RNA, nucleotides) absorb light nucleic acid (DNA, RNA, nucleotides) absorb light

at 260nmat 260nm protein absorbs light at 280 nm 230nm: protein absorbs light at 280 nm 230nm:

guanidineguanidine► A260/A280 ratio is a measure of DNA purity A260/A280 ratio is a measure of DNA purity ► The absorbance wavelength is directly The absorbance wavelength is directly

proportional to the concentration of nucleic proportional to the concentration of nucleic acid.acid.

Determining ConcentrationDetermining Concentration►Formula Formula

Concentration = 260 Reading * Concentration = 260 Reading * Absorbance unit * Dilution factor Absorbance unit * Dilution factor

►One optical density or absorbance unit One optical density or absorbance unit at 260 nm is equal to at 260 nm is equal to 50 ug/mL for DNA50 ug/mL for DNA 40 ug/mL for RNA.40 ug/mL for RNA.

►Textbook page 77 has sample Textbook page 77 has sample calculations.calculations.

Determining PurityDetermining Purity► The OD at 260nm should be 1.6-2.00 times more The OD at 260nm should be 1.6-2.00 times more

than the absorbance at 280nm.than the absorbance at 280nm.► Divide the OD at 260nm by the OD at 280nm to get Divide the OD at 260nm by the OD at 280nm to get

the ratio.the ratio.► If the 260nm/280nm ratio is less than 1.6 for DNA, If the 260nm/280nm ratio is less than 1.6 for DNA,

2.0-2.3 for RNA this indicates contamination, 2.0-2.3 for RNA this indicates contamination, usually with protein.usually with protein.

► DNA -If the OD ratio is higher than 2.0 it may be DNA -If the OD ratio is higher than 2.0 it may be contaminated with RNA.contaminated with RNA.

► Ratio of the readings : O.D.260/O.D.280 is a Ratio of the readings : O.D.260/O.D.280 is a measure of purity. measure of purity.

► Pure preparations of DNA and RNA have O.D Pure preparations of DNA and RNA have O.D 260/280 of 1.8 and 2.0 respectively. 260/280 of 1.8 and 2.0 respectively.

FluorometryFluorometry► Fluorometry utilizes fluorescent dyes which specifically

bind DNA or RNA. ► It requires a negative control (to set the zero point on the

fluorometer) and a standard of known concentration. ► The fluorometer shines light on the sample (excitation)

and then measures level of fluorescent light being emitted to the side (at a 90º angle) of the excitation light beam.

► The fluorescent dyes are relatively specific to nucleic acids as opposed to protein and other cellular components.

► The fluorescence of the dyes increases when they bind nucleic acids.

Fluorometry► Fluorometry is about 1,000x more sensitive than

spectrophotometric absorbance (i.e. measurement of A260) and less susceptible to protein and RNA contamination.

► However it also does not give a crude measurement of purity (like an A260/A280 ratio) nor does it assure that the DNA or RNA is not degraded (e.g. like size determination by gel electrophoesis).

► Do not use glass (spectrophotmetry) cuvettes in a fluorometer because the frosted glass on the side of the cuvette interferes with detection of fluorescent light.