Nucleic Acid Extraction Methods

8/12/2019 Nucleic Acid Extraction Methods

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Unit 6 Nucleic Acid

Extraction MethodsTerry Kotrla, MS, MT(ASCP)BB

Fall 2007


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Purpose

To release nucleic acid from the cell for usein other procedures

Must be free from contamination withprotein, carbohydrate, lipids or other nucleicacids.

Used pure nucleic acids for testing.


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Isolation

Routinely isolated from human, fungal,bacterial and viral sources.

Pretreat to make nucleated cells available,

whole blood

Tissue samples

Microorganisms

Need sufficient sample for adequate yield.


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Organic Isolation

Must purify DNA by removing contaminants.

Accomplished by using combination of highsalt, low pH and an organic mixture ofphenol and chloroform.

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


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Phenol/Chloroform

Biphasic emulsion forms

Hydrophobic layer on bottom has celldebris.

Hydrophilic layer on top has dissolved DNA

Remove top layer, add cold ethanol, DNA

precipitates out.


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Inorganic Isolation Methods

Also called salting out. Uses low pH and high salt condition to selectively

precipitate proteins.

DNA is left in solution (picture on far left).

Precipitate out DNA with isoproproanol (middle and rightside pictures).

I know this is not scientific but the precipitated out DNA isusually referred to as snotty.


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Solid Phase Isolation

More rapid and effective

Use solid matrix to bind the DNA.

Wash away contaminants.

Elute DNA from column

Textbook page 70, Figure 4.3


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Solid Phase Isolation

The diagram below explains the attractiveproperties of solid phase for DNA and RNA.


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Crude Lysis

Used for:

Screening large numbers of samples

Isolation of DNA in limited amounts

Isolation from challenging samples, ie, paraffinembedded tissue

Usually not done in clinical laboratory.

Textbook page 71, Figure 4-4


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Isolation of Mitochondrial DNA

Mitochondrial DNA is passed fromgeneration to generation along the maternallineage.

Centrifugation to separate out

Lyse

Precipitate with cold ethanol.


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Isolation of RNA

Requires STRICT precautions to avoidsample degradation.

RNA especially labile.


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RNAses

RNases are naturally occurring enzymes thatdegrade RNA

Common laboratory contaminant (frombacterial and human sources)

Also released from cellular compartmentsduring isolation of RNA from biologicalsamples

Can be difficult to inactivate


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RNAses

RNAses are enzymes which are smallproteins that can renature and becomeactive.

MUST be eliminated or inactivated BEFOREisolation.

CRITICAL to have a separate RNAse freearea of lab.


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Protecting Against RNAse

Wear gloves at all times

Use RNase-free tubes and pipet tips

Use dedicated, RNase-free, chemicalsPre-treat materials with extended heat (180

C for several hours), wash with DEPC-

treated water, NaOH or H2O2Supplement reactions with RNase inhibitors


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Total RNA

80-90% of total RNA is ribosomal RNA.

2.5-5% is messenger RNA


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Organic RNA Extraction

1. Lyse/homogenize cells2. Add phenol:chloroform:isoamyl

alcohol to lysed sample, andcentrifuge

3. Organic phase separates fromaqueous phase

Organic solvents on bottom Aqueous phase on top (contains

total RNA) Cellular debris and genomic DNA

appears as a film of debris at theinterface of the two solutions

4. Remove RNA solution to a clean

tube; precipitate RNA and washwith ethanol, then resuspend RNAin water


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Isolation of PolyA (messenger) RNA

Only 2.5-5%

mRNA molecules have a tail of As at the 3end (polyA tail)

Oligo(dT) probes can be used to purifymRNA from other RNAs

mRNA can be eluted from oligo(dT) matrixusing water or low-salt buffer

Textbook page 75, Figure 4.7


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Electrophoresis

Analyze DNA and RNA for quality byelectrophoresis.

Fluorescent dyes

Ethidium bromide

SybreGreen

Appearance depends on type of DNAisolated.

Will be covered in more detail in unit 7.


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Staining with Ethidium Bromide andSybr Green

DNA is electrophoresed and stained. LeftEthidium Bromide RightSybr Green

The lane in the left side of the picture on the left is aladder which has fragments of knownbase pair (bp)sizes. This allows determination of the size of isolatedfragments.


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Spectrophotometry

Sample absorbances are determined on thespectrophotometer at 260nm and 280nm

nucleic acid (DNA, RNA, nucleotides) absorb light at

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

A260/A280 ratio is a measure of DNA purity

The absorbance wavelength is directlyproportional to the concentration of nucleic acid.


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Determining Purity

The OD at 260nm should be 1.6-2.00 times more than theabsorbance at 280nm.

Divide the OD at 260nm by the OD at 280nm to get theratio.

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

contaminated with RNA. Ratio of the readings : O.D.260/O.D.280 is a measure of

purity. Pure preparations of DNA and RNA have O.D 260/280 of1.8 and 2.0 respectively.


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Fluorometry

Fluorometry utilizes fluorescent dyes which specifically bindDNA or RNA.

It requires a negative control (to set the zero point on thefluorometer) and a standard of known concentration.

The fluorometer shines light on the sample (excitation) andthen measures level of fluorescent light being emitted tothe side (at a 90 angle) of the excitation light beam.

The fluorescent dyes are relatively specific to nucleic acidsas opposed to protein and other cellular components.

The fluorescence of the dyes increases when they bindnucleic acids.


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Fluorometry

Fluorometry is about 1,000x more sensitive thanspectrophotometric absorbance (i.e. measurement ofA260) and less susceptible to protein and RNAcontamination.

However it also does notgive a crude measurement ofpurity (like an A260/A280 ratio) nor does it assure that theDNA or RNA is not degraded (e.g. like size determinationby gel electrophoesis).

Do not use glass (spectrophotmetry) cuvettes in a

fluorometer because the frosted glass on the side of thecuvette interferes with detection of fluorescent light.

Nucleic Acid Extraction Methods

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