Molecular Biology, Exam 1, DNA Isolation

DNA can persist long after cells and tissues have decomposed, allowing its recovery from…

crime scenes, archealogical remains, ancient / frozen environment

why is it possible to isolate DNA from cells and recover it long after cellular components have degraded

DNA’s chemical stability

what can damage and degrade DNA

high temp and UV exposure, high amt of microbial activity

Why is DNA chemically strong, but physically weak

• high molecular weight, with long, thin molecules that are easily broken by mechanical forces

  • the longer the molecule, the more susceptible it is to shearing

• hydrodynamic shearing - DNA can break when solutions are rapidly pipetted, vortexed, passed through narrow openings

DNA sonication

• high energy waves go through sample to open up DNA

• longer sonication time = higher risk of DNA damage

• breaks DNA into smaller pieces

all DNA isolation methods rely on…

1. disruption / lysis of cellular membrane (open up cell membrane)

2. removal / inactivation of proteins and other macromolecules of the cell (removal of cellular debris)

3. precipitation and recovery of DNA (refinement of sample)

goals of DNA isolation

• separate DNA from other cellular components (proteins, RNA, lipids, cellular debris)

• Preserve the DNA in an intact form (minimize shearing and prevent degradation by endogenous nucleases)

• Inactivate endogenous nucleases (heat can denature / inactivate nucleases and EDTA chelates Mg 2+ and other divalent cations required by many DNases)

  • 95-100 C = kills nucleases

  • EDTA “grabs” onto divalent (+2 charge) on ions to remove them; also removes Mg 2+, a cofactor to nucleases (protective agent)

DNA Isolation - Cell Lysis

use of detergent (SDS = Sodium dodecyl sulfate) to solubilize the lipid bilayer of the cell membrane (phospholipid bilayer)

Separating DNA from other cellular components

• DNA must be separated from proteins and other cellular components

• DNA separation methods: organic extraction and salt precipitation (“salting out”)

Organic Extraction - Traditional Method Basics

• phenol and chloroform are used to separate DNA from proteins and other cellular components → one of the first published methods

• when phenol and chloroform is mixed with the cellular lysate, two phases form

  • upper aqueous phase: DNA remains primarily in this phase

  • lower organic phase: denatured proteins and lipids partition into this phase

• phenol denatures proteins and promotes their removal from the aqueous DNA-containing phase

Organic Extraction - Traditional Method Steps

• Phase separation: after cell lysis, phenol-chloroform mixture is added. Phenol is an organic solvent, and chloroform is added to enhance the separation of phases

• when the sample is mixed and centrifuged the following occurs:

  • aqueous phase: the DNA, being hydrophilic (water lovign) remaind in the aqueous phase

  • interface: transition zone between aqueous and organic phase; contains denatured proteins and lipids

  • organic phase: the phenol and chloroform (which are both hydrophobic) denatures, causing them to migrate into the organic phase (bottom layer)

Salting Out Methodology - Basics

• at high salt concentration, proteins become denatured and lose solubility, causing the denatured proteins to precipitate (the shape changes and clumps together)

• common salts: sodium acetate, potassium acetate, sodium chloride

  • the acetates act as a weak acid to balance pH

• precipitated proteins are removed by centrifugation

• DNA remains in the solution, in the upper aqueous phase of the preparation after centrifugation

Salting Out Methodology - the process

1. after cell lysis, a high concentration of salt (sodium acetate) is added to the lysate. the high ionic strength of the solution affects the solubility of proteins in aqueous solution

2. proteins - for many, composition includes charged amino acids; in the presence of high salt concentration, the salt ions (Na+) neutralize the charges on the proteins’ surfaces. Reduces the proteins solubility, causing the proteins to aggregate or “salting out” of solution

3. DNA - does not precipitate in the high salt solution. It remains in the aqueous phase this stage

salting out methodology - step by step

1. cell lysis

2. protein digestion (use of proteinase K)

3. protein precipitation via high salt concentration

4. centrifugation to remove the proteins

5. supernatant will contain the desired DNA

6. DNA is precipitated by the addition of an alcohol

7. DNA re-suspended in optimal buffer (or water)

precipitation of DNA

• ethanol (100-96%) or isopropyl is typically used to layer on the top of concentrated solution of DNA

• strands of DNA can be spooled with a sterile glass rod

• most commonly, the mixture of ethanol / DNA solution is subjected to centrifugation (results in the formation of DNA pellet)

• DNA sample can then be”washed” to help remove residual salts / organic carryovers

  • “desalting” of DNA can be accomplished using 70% ethanol preparation

• help to remove any remaining salt or contaminants and concentrates DNA

steps of DNA precipitation

• step helps to remove any remaining salt or contaminants, and concentrates the DNA

• alcohol is added to the aqueous phase, and the mixture is centrifuged again; the DNA will form a visible pellet at the bottom of the tube

• the pellet may be washed with alcohol (usually ethanol) to remove residual salts, and then the DNA is air dried and resuspended in an appropriate solvent (TE buffer and nuclease-free water)

Why does the salting out method work?

• proteins have solubility properties that are influenced by high salt concentrations. At higher ionic solubility, their solubility decreases, and they precipitate out of aqueous solution

• DNA remains soluble because of its charge (sugar-phosphate backbone) making it highly soluble in water, event in the presence of salt. High salt does not effect the solubility of DNA in the same way it affects proteins

what do typical commercial DNA kits comprise of

• sodium chloride / sodium acetate

• tris base (trimethamine) - buffer to maintain optional pH

• EDTA - binds and removes metal ions

• SDS - detergent

• enzymes to remove proteins and/or RNA: Rnases and Proteinase K

spectrometry to measure DNA concentration

• DNA concentration can be measured by absorbance of specific wavelengths of energy via a spectrophotometer

• measuring the absorbance of the solution at 260 nm and 280 nm is common for the determination of DNA concentration at 260 nm (as well as 280 nm)

• expressed in units of micrograms of DNA / mililiter or given by micrograms / microliter

• could show purity of DNA in comparison of 260nm/280nm ratio

  • a ratio of 1.7-1.9 is considered optimal

assessing integrity of DNA

• running a small amount of the sample through an agarose gel is common method of assessing integrity of DNA

• can detect degradation of DNase activities and/or mechanical sheering of DNA that occurred during the extraction protocols

• high quality of genomic DNA should migrate slowly (due to high molecular weight) as single bands, with little to no smearing

why is it important to check the quality of DNA isolation

• the product of DNA extractions is used in subsequent experiments

• poor quality DNA will not preform well for RE digestion or PCR

• without good starting materials, all other analyses are not valid