Unit 6 Nucleic Acid Unit 6 Nucleic Acid Extraction MethodsExtraction MethodsTerry Kotrla, MS, MT(ASCP)BBTerry Kotrla, MS, MT(ASCP)BB

Fall 2007Fall 2007

PurposePurpose

►►To release nucleic acid from the cell for use To release nucleic acid from the cell for use in other proceduresin other procedures

►►Must be free from contamination with Must be free from contamination with protein, carbohydrate, lipids or other nucleic protein, carbohydrate, lipids or other nucleic acids.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.

►►PretreatPretreat to make nucleated cells available,to make nucleated cells available,whole bloodwhole bloodTissue samplesTissue samplesMicroorganismsMicroorganisms

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

Organic IsolationOrganic Isolation

►►Must purify DNA by removing contaminants.Must purify DNA by removing contaminants.►►Accomplished by using combination of high Accomplished by using combination of high

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

►►To avoid RNA contamination add To avoid RNA contamination add RNAseRNAse, , enzyme that degrades RNA.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 DNAHydrophilic layer on top has dissolved DNA►►Remove top layer, add cold ethanol, DNA Remove top layer, add cold ethanol, DNA

precipitates out.precipitates out.

Inorganic Isolation MethodsInorganic Isolation Methods►► Also called Also called ““salting outsalting 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 Precipitate out DNA with isoproproanolisoproproanol (middle and right (middle and right

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

usually referred to as usually referred to as ““snottysnotty””..

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 Crude LysisLysis

►►Used for:Used for:Screening large numbers of samplesScreening large numbers of samplesIsolation of DNA in limited amountsIsolation of DNA in limited amountsIsolation from challenging samples, Isolation from challenging samples, ieie, paraffin , paraffin embedded tissueembedded tissue

►►Usually not done in clinical laboratory.Usually not done in clinical laboratory.►►Textbook page 71, Figure 4Textbook page 71, Figure 4--44

Isolation of Mitochondrial DNAIsolation of Mitochondrial DNA

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

►►RNasesRNases are naturally occurring enzymes that are naturally occurring enzymes that degrade RNAdegrade RNA

►►Common laboratory contaminant (from Common laboratory contaminant (from bacterial and human sources)bacterial and human sources)

►►Also released from cellular compartments Also released from cellular compartments during isolation of RNA from biological during isolation of RNA from biological samples samples

►►Can be difficult to inactivateCan be difficult to inactivate

RNAsesRNAses

►►RNAsesRNAses are enzymes which are small are enzymes which are small proteins that can proteins that can renaturerenature and become and become active.active.

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

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

Protecting Against Protecting Against RNAseRNAse

►►Wear gloves at all timesWear gloves at all times►►Use Use RNaseRNase--free tubes and free tubes and pipetpipet tipstips►►Use dedicated, Use dedicated, RNaseRNase--free, chemicalsfree, chemicals►►PrePre--treat materials with extended heat (180 treat materials with extended heat (180

C for several hours), wash with DEPCC for several hours), wash with DEPC--treated water, treated water, NaOHNaOH or H2O2or H2O2

►►Supplement reactions with Supplement reactions with RNaseRNase inhibitorsinhibitors

Total RNATotal RNA

►►8080--90% of total RNA is ribosomal RNA.90% of total RNA is ribosomal RNA.►►2.52.5--5% is messenger RNA5% is messenger RNA

Organic RNA ExtractionOrganic RNA Extraction1.1. LyseLyse/homogenize cells/homogenize cells2.2. Add Add phenol:chloroform:isoamylphenol:chloroform:isoamyl

alcohol to alcohol to lysedlysed sample, and sample, and centrifugecentrifuge

3.3. Organic phase separates from Organic phase separates from aqueous phaseaqueous phase

Organic solvents on bottomOrganic solvents on bottomAqueous phase on top (contains Aqueous phase on top (contains total RNA)total RNA)Cellular debris and genomic DNA Cellular debris and genomic DNA appears as a appears as a ““filmfilm”” of debris at the of debris at the interface of the two solutionsinterface of the two solutions

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

Affinity Purification of RNAAffinity Purification of RNA

1.1. LyseLyse cells, and spin to remove cells, and spin to remove large particulates/cell debrislarge particulates/cell debris

2.2. Apply Apply lysatelysate (containing nucleic (containing nucleic acids and cellular contaminants) to acids and cellular contaminants) to column with glass membranecolumn with glass membrane

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

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

Isolation of Isolation of PolyAPolyA (messenger) RNA(messenger) RNA

►►Only 2.5Only 2.5--5%5%►►mRNA molecules have a tail of AmRNA molecules have a tail of A’’s at the 3s at the 3’’

end (end (polyApolyA tail)tail)►►Oligo(dTOligo(dT) probes can be used to purify ) probes can be used to purify

mRNA from other mRNA from other RNAsRNAs►►mRNA can be eluted from mRNA can be eluted from oligo(dToligo(dT) matrix ) matrix

using water or lowusing water or low--salt buffersalt 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 dyesEthidiumEthidium bromide 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 Staining with EthidiumEthidium Bromide and Bromide and SybrSybr Green Green

►► DNA is DNA is electrophoresedelectrophoresed and stained. and stained. ►► Left Left –– EthidiumEthidium Bromide Right Bromide Right –– SybrSybr GreenGreen►► The lane in the left side of the picture on the left is a The lane in the left side of the picture on the left is a

““ladderladder”” which has fragments of which has fragments of knownknown base pair (base pair (bpbp) ) sizes. This allows determination of the size of isolated sizes. This allows determination of the size of isolated fragments.fragments.

SpectrophotometrySpectrophotometry

►► Sample Sample absorbancesabsorbances are determined on the are determined on the spectrophotometer at 260nm and 280nmspectrophotometer at 260nm and 280nm

nucleic acid (DNA, RNA, nucleotides) absorb light at nucleic acid (DNA, RNA, nucleotides) absorb light at 260nm260nmprotein absorbs light at 280 nm 230nm: guanidineprotein absorbs light at 280 nm 230nm: guanidine

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

Determining ConcentrationDetermining Concentration

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

►►One optical density or absorbance unit at One optical density or absorbance unit at 260 nm is equal to 260 nm is equal to

50 50 ug/mLug/mL for DNAfor DNA40 40 ug/mLug/mL for RNA.for RNA.

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

Determining PurityDetermining Purity

►► The OD at 260nm should be 1.6The OD at 260nm should be 1.6--2.00 times more than the 2.00 times more than the absorbance at 280nm.absorbance at 280nm.

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

►► If the 260nm/280nm ratio is less than 1.6 for DNA, 2.0If the 260nm/280nm ratio is less than 1.6 for DNA, 2.0--2.3 2.3 for RNA this indicates contamination, usually with protein.for RNA this indicates contamination, usually with protein.

►► DNA DNA --If the OD ratio is higher than 2.0 it may be 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 measure of Ratio of the readings : O.D.260/O.D.280 is a measure of purity. purity.

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