DNA ISOLATION

Dna isolation

Fundamental process in molecular biology that involves separating deoxyribonucleic acid from other cellular components such as proteins, RNA and carbohydrates

Proteinase K

Protease added during the lysis step to digest cellular proteins, remove histones bound to DNA and inactivate endogenous nucleases that could otherwise degrade the target DNA

Phenol-chloroform-isoamyl

Classic liquid-liquid extraction technique used to isolate high-purity, high-molecular-weight DNA by separating nucleic acids from proteins, lipids, and cellular debris

25:24:1

Phenol-chloroform-isoamyl ratio

Salting out

Another term for inorganic method of DNA isolation

70% ethanol (cold)

In inorganic dna isolation, what is used to wash to purify the precipitated DNA pellet?

pH

DNA placement during extraction depends heavily on what?

Top layer

In an alkaline/neutral pH (7-8), dna is found where?

Bottom layer

In an acidic pH (4.5-5.0), dna is found where?

White blood cell

Common source of genomic DNA from whole blood

1. Sample collection

2. Cell lysis

3. Removal of contaminants

4. DNA precipitation

5. DNA purification

6. DNA rehydration

Steps in DNA isolation

Tissue

Fresh, frozen or paraffin embedded specimen

Microorganisms

Used for pathogen identification

Erythrocytes (RBC)

Cell that does not contain nuclear dna

Cell lysis

Breaking open the cell wall, cell membrane and nuclear membrane using physical force or chemical/enzymatic agents

Precipitation

Removing cellular debris and proteins, often by centrifugation and alcohol precipitation

Purification/elution

Washing and resuspending the isolated DNA in a safe buffer for downstream applications like PCR or sequencing

Dna purification

Micro-process of separating and cleaning DNA from cellular components like proteins, lipids and RNA after breaking open the cells

Lysate

Goal of lysis is to rapidly and completely disrupt cells in a sample to release nucleic acid into what?

Clearing of lysate

Cellular lysates may need to have cellular debris removed prior to nucleic acid to reduce carryover of unwanted materials into the purification method

Silica spin column or magnetic beads

Dna binds to a solid matrix such as a what?

Bind capacity

Indication of how much nucleic acid an isolation chemistry can bind before it reaches the capacity of the system and no longer isolates more of that nucleic acid

Alcohol

Wash buffers are generally what?

Eluction

Purified, concentrated DNA is then released from the matrix into a clean, low-salt buffer or water

Organic reagent

Main principle of organic method

Salt precipitation

Main principle of inorganic method

Magnetic beads and silica column

Main principle of solid phase

Boiling

Main principle of simple method

Phenol and chloroform

Common reagent/material of organic method

Sodium chloride and ammonium acetate

Common reagent/material of inorganic method

Silica membrane and magnetic beads

Common reagent/material of solid phase

Chelex

Common reagent/material of simple method

Organic method

Best DNA isolation method

Inorganic

Not that expensive but has lower purity of DNA isolation

Solid-phase method

Routine hospital PCR laboratory

Simple method

Student teaching laboratory

Organic method

DNA sequencing research

Solid-phase method

Automated exctration system

Solid phase method

DNA isolation method for quick and rapid, highly reproducible extraction method

Organic method

A research laboratory requires very high quality genomic DNA for whole genome sequencing and is willing to use more labor intensive procedures