MOLBIO LEC NUCLEIC ACID EXTRACTION METHODS

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

Must be free from contamination with protein, carbs, lipids, or other nucleic acids

Used pure nucleic acids for testing

Purpose of nucleic acid extraction (3)

Isolation

routinely isolated from human, fungal, bacterial, and viral sources

Pretreatment

ex. wash 3 times

Whole blood
Tissue samples
Microorganisms

What specimen needs pretreatment to make nucleated cells available? (3) WTM

Organic isolation

isolation using organic components

Phenol or chloroform

2 commonly used organic isolators

RNAse

to avoid RNA contamination, this enzyme that degrades RNA is added

Phenol/Chloroform

biphasic emulsion forms

Hydrophobic or hydrophilic

Biphasic

cell debris

Hydrophobic layer on bottom has:

Dissolved DNA

hydrophilic layer on top has:

Cold ethanol

What to add so that DNA precipitates out?

Inorganic isolation method

uses low pH and high salt condition for selectively precipitate proteins

Salting out

other name for inorganic isolation methods

Snotty

the precipitated DNA is usually referred to as:

Solid phase isolation

more rapid and effective
use solid matrix to bind DNA
Wash away contaminants
Elute DNA from column

Crude lysis

used for screening large numbers of samples

isolation of DNA in limited amounts

isolation from challenging samples ex. paraffin embedded tissue

usually not done in clinical lab

Mitochondrial DNA

is passed from generation to generation along the maternal

Yes

is RNAse difficult to inactivate?

Common laboratory contaminant

RNAse is a common:

Have a separate RNAse free area of lab

It is critical to:

180 degrees Celsius for several hours

Pretreatment with extended heat how hot and how long?

80-90%

Percent of total RNA is rRNA

2.5-5%

Percent is messenger RNA

Lyse cells
Add phenol, chloroform, isoamyl alcohol to lysed sample and centrifuge
Organic phase separates from aqueous phase
Remove RNA solution to a clean tube: precipitate RNA and wash with ethanol

Steps for organic extraction

poly A tail

mRNA molecules have a tail of A's at the 3' end

OligodT

probes that can be used to purify mRNA from RNA

Electrophoresis

analyzes DNA and RNA for quality

Ethidium bromide and SybreGreen

2 Fluorescent dyes for electrophoresis (EthBroSybGreen)

Ladder

lane which has fragments of known base pair sizes

Spectrophotometry

determines concentration of DNA and RNA

260 Reading * Absorbance unit * Dilution factor

Concentration formula

260-280nm

50 micrograms per milliliter

260 nm is equal to how much for DNA?

40 micrograms per milliliter

260 nm is equal to how much for RNA?

280nm

protein absorbs light at:

230nm

Guanidine absorbs light at:

260nm

Nucleic acid absorbs light at:

1.8

Pure preparations of DNA/RNA at 260 OD

2

Pure preparations of DNA/RNA at 280

Fluorometry

utilizes fluorescent dyes which specifically bind DNA or RNA.

Because the frosted glass of the cuvette interferes with detection of fluorescent light

Why do we not use glass cuvettes in fluorometry?