Nucleic Acid Extraction Methods

What sources can nucleic acids be extracted from?

Human, fungal, bacterial, and viral sources

Where are nucleic acids most commonly isolated from in the clinical lab?

peripheral blood

Why us peripheral blood used most often to isolate nucleic acids?

• not very invasive

• less prone to contamination

• gives better yield than buccal swabs

Why does peripheral blood give a better yield than buccal swabs?

it has more nucleated cells (dependant on WBC count)

What is the ideal specimen when isolating nucleic acids?

anti-coagulated whole blood, using EDTA

• store for 7 days at RT

• store for longer in aliquots at -70

Why must heparin be avoided as an anticoagulant?

it can inhibit the action of DNA polymerases

Sample preparation: Whole blood and bone marrow aspirates

WBCs purified of RBCs - purification can be done via density gradient or differential lysis

Why is serum not a good source of nucleic acids?

Because all the WBCs are in the clot - serum is acellular

Sample preparation: Plasma

• contain free WBCS carrying nucleic acids and cell-free nucleic acids (circulating nucleic acids)

• collected by centrifugation

Sample preparation: Tissue

• fresh/frozen - grinding and homogenizing issue

• fixed/embedded - de-paraffinized with xylene

Sample preparation: Bacteria/Fungi/Viruses

May require additional preparations. Ex. gram + bacteria requires extra steps to break through thick cell wall

What is the purpose of the purification step?

to remove contaminating proteins, lipids, carbohydrates, and cell debris

Organic isolation

combination of low-pH, high salt, and organic mixture of phenol and chloroform which readily dissolves hydrophobic contaminants such as lipids, lipoproteins, cell debris, and most DNA-associated proteins

Inorganic isolation

uses low-pH, high salt conditions to precipitate proteins leaving DNA in solution; then uses ethanol to precipitate the DNA. Also knows as “salting out”.

Solid phase isolation

more rapid and comparably effective is the use of solid matrices including beads and columns to bind and wash the DNA. Solid phase isolation is used for several automated DNA isolation systems that lyse cells, isolate and elite DNA automatically

What happens during the cell lysis step of DNA organic isolation?

• SDS creates holes in cell membrane by disrupting the protein bonds

• NaOH loosens the cell wall by disrupting hydrogen bonds

• NaOH is alkaline this increase the solutions pH

What happens during the acidification step of DNA organic isolation?

1. Acetic acid and salt returns pH to neutral

2. Salts in NaOH bind to any free protein structures removing unwanted materials

3. Time sensitive step - pH too high for too long destabilizes DNA

What does SDS stand for?

Sodium dodecyl sulphate

What happens if the pH is too high for too long during the lysis step?

DNA can destabilize d/t sugar backbone breakdown

What happens if pH drops too low during the acidification step?

DNA is prone to depurination - loss of purine bases A & G

What happens if pH is left too high during the acidification step?

the efficacy of the extraction step may be affected

What happens during the extraction step of DNA organic isolation?

Phenol is used for extraction, it will sink to the bottom of the tube collecting impurities and the DNA will remain bound to water at the top of the tube

Phenol is not an alcohol, what is it?

a solvent

What happens during the precipitate step of DNA organic isolation?

DNA is washed 1-2 times with ethanol to remove the salt contamination form the extraction step

What are the cons of DNA organic isolation?

• Time consuming

• Potential for contamination or sample mix-ip

• Phenol is toxic

What is the main difference between organic and inorganic isolation?

Inorganic uses salts where as Organic uses Phenol

Inorganic isolation is also know as?

“salting-out”

What happens during the cell lysis phase of inorganic DNA isolation?

1. TE buffer is used to solubilize DNA, while protecting it from degradation

2. SDS breaks lipid membrane from the cell exterior

What happens during the precipitation step of inorganic DNA isolation?

1. Precipitate proteins with sodium acetate which has a lower pH and is a salt

2. Precipitate DNA with ethanol or isopropanol

   1. remove excess salts with alcohol wash

What happens during the re-suspend step of inorganic DNA isolation?

DNA is re-suspended with TE buffer or H2O

What are the cons of Inorganic DNA isolation?

• time consuming

• most prone to salt contamination downstream - can inhibit PCR if not removed with alcohol washes

Why is solid phase isolation the choice of most labs?

it is easily automated

What are the cons of solid phase isolation?

• more expensive

• comes in a kit - ends in waste is short one item

Why is RNA hard to isolate?

it is unstable, time-sensitive, and hard to store long term

What blood tube should RNA isolation samples be collected in?

Grey top tubes - sodium oxalate

How should purified RNA be stored?

• stored as ethanol precipitates at -70 degrees.

• the number of freeze/thaw cycles should be minimized for specimens and solutions

What is done to prevent RNase contamination in the lab?

• Separate lab area designated RNase free

• Disposable/reagents - RNase free

• Reactions - add RNase inhibitors

What is the most abundant type of RNA in cells?

rRNA - 80-90%

• made of large and small subunits that can be separate isolates during visualization if the RNA is good quality

What type of RNA are we trying to extract and how of of the cell does it make up?

mRNA - 2.5-5%

How do we enrich the yield of mRNA?

single stranded oligomers of thymine/uracil are immobilized on a matrix resin column. The addition of T/U to the matrix will bind with the polyA tail found exclusively on mRNA and trap the molecules.

What are the cons of Organic RNA isolation?

• RNA contamination with RNase is very common

• easy to cause patient mix up

• phenol is toxic

Why cant we use high pH alkaline solutions for RNA (Salting out/ inorganic isolation)?

it causes hydrolysis (RNA strands denatures and splits into fragments) leaving us with a fragmented product and can cause issues with replication in vitro when trying to analyze

What does chaotropic mean?

“chaos forming” - disrupts the ability of hydrogen bond to occur and encourages RNA to absorb onto the matrix

Electrophoresis

use of dyes to stain DNA or RNA and run on an agarose gel

Spectrophotometry

measure the absorbance of light by a DNA sample at specific wavelengths, typically around 260 nm, which is the peak absorbance for nucleic acids.

What is the absorptivity constant for DNA?

50ug/ml

What is the absorptivity constant for RNA?

40ug/ml

Fluorometry

measure the fluorescence emitted by a DNA sample after it is exposed to specific wavelengths of light. Dyes insert themselves between hydrogen bonds in dsDNA backbone.

What does a higher absorbance value indicate?

higher yield

How much sample is required for spectrophotometry?

2ul

What is the disadvantage of spectrophotometry?

it is unable to fully distinguish between double and low quality single stranded DNA

What does fluorometry require that spectrophotometry doesn’t?

calibration - create a linear comparison with a low and high standard

What is the advantage of fluorometry?

It is able to differentiate between low quality sheared DNA and high quality dsDNA by the use of an intercalating dye

Assessing DNA with electrophoresis

• tight groupings mea DNA fragments are of smaller size/characteristics

• multiple more faint bands mean fragmented DNA

What is DNAs purity range?

1.6-2

Assessing spectrophotometry

• values less than 1.6 indicate there may be contamination with unacceptable amounts of proteins or salts

• values >2 indicate contamination with residual phenols

Assessing RNA with electrophoresis

• high quality RNA should show with 2 bands

  • one representing 28s (large subunit)

  • one representing 18s (small subunit)

• May see faint banding of mRNA

What is RNAs purity ratio?

2.0-2.3