G-DNA Extraction, Quantitation, Purity and Quality

ppt: DNA extraction theory + DNA quantitation, purity and quality theory

Types of specimens

whole blood : collected in EDTA vacutainer (can use yellow top), transport and store at room temp for up to 4 days

buffy coat (fresh or frozen) : fresh at room temp up to 4 days or frozen at -80oC for years

guthrie spot

• from a newborn heel prick onto a piece of paper

tissue (fresh, frozen or paraffin embedded) : fresh at 4oC for 2 days, -80oC for years, paraffin indefinitely

Saliva (if in preserving fluid) : for years at room temp

amniotic fluid : pellet stored at -80 for years

cultured cells : up to 8 weeks with subculturing

bone marrow

explain paraffin embedded samples

can be stored at romm temp

tissue is removed from body, fixed, dehydrated, infiltrated with molten paraffin wax, embedded into a block, sectioned (cut and placed on a slide) and then stained 

preserves cells, tissue components and architecture 

pathologists view stained tissu on slide to analyze

DNA can be extracted for molecular analysis

uses formalin as fixative but it degrades DNA the longer its on tissue

• DNA extracted is often poor 

• only used for tests that require low molecular weight DNA

need pre-treatment before use of DNA to remove paraffin

• soak in xylene to dissolve paraffin

• heat sample to min 70oC to melt paraffin

general requirements for DNA extraction 

lysis of the cell and nuclear membranes 

removal of proteins and cellular debris 

alcohol precipitation of DNA

additional steps depend on specimen type 

common extraction methods 

organic (phenol/chloroform)

column (qiagen)

salt (puregene)

magnetic bead

chelex

steps for organic method

1. initial sample processing (depends on type)

   1. for blood:

      1. lyse RBC with ammonium chloride-tris

      2. spin to remove debris and rinse WBCs with isotonic saline

2. lyse cell/nuclear membranes (with SDS in presence of tris EDTA)

   1. SDS = detergent used to dissolve lipid membranes and bind to/denature/solubilize proteins to disassociate them from DNA

   2. tris =buffer to resist pH change

   3. EDTA chelates Mg ions → enzymes (nucleases) inactivated

3. digest proteins with proteinase K (still active after step 2)

4. phenol extraction

   1. dissolves proteins and other organic solutes (and denatures proteins)

   2. DNA remains dissolved in upper aqueous layer (close to interphase) after spinning.

   3. lower organic layer

5. phenol/chloroform extraction

   1. second phenol step to remove remaining organics

   2. chloroform:isoamylalcohol (24:1)

      1. chloroform: dissolves phenol to remove from DNA

      2. iso: reduces foaming 

6. chloroform exraction

   1. dissolve residual phenol (could precipitate with DNA and cause degradation

7. salt (optional): 3M sodium acetate 

   1. added to aqueous DNA to help it precipitate by making it less soluble in water 

   2. used when need max yield

8. alcohol precipitate DNA

   1. add ice cold ethanol (95%) or isopropanol

      1. use isoprop when small volume of aqueous DNA or when adding salt (prevents salt from precipitating in the cold)

   2. invert alot until visible DNA that looks like cotton precipitate

9. 70% ethanol wash (if necessary)

   1. dissolves salt but not DNA

   2. use if salt was added 

10. dissolve DNA

steps for column method (qiagen)

1. protease added directly to sample 

2. lysis buffer immediatly after 1.

   1. contains guanidine hydrochloride (chaotropic salt) to denature proteins

   2. and SDS todissolve lipid membranes and binds to/denatures/solubilizes proteins

   3. at a specific pH and is high salt

3. incubate (at right temp for enz)

4. add EtOH to precipitate DNA in solution

5. load sample into column containing silical gel membrane 

   1. DNA binds to membrane, organics flow through 

6. add waash buffer and spin (repeated several times)

   1. buffers contain EtOH and salt to keep DNA precipitated 

   2. at correct pHo bind DNA tp membrane 

   3. purpose = wash away contaminants and cellular material 

7. elute/dissolve pure DNA off column (spin to collect in tube 

   1. solution used at correct pH and low salt 

what is a chaotropic salt 

it destabilizes H bonds, van der Waals forces and hydrophobic interactions 

leads to destabilization of proteins 

• important to degrade DNases

steps for salt extraction

1. lysis of RBCs and spin to pellet WBC

2. lysis of WBC and nuclear membranes with SDS-TE buffer

3. add high salt buffer

   1. causes proteins to coagulate = thick and heavy → precipitate out of solution

4. spin (DNA in supernatant)

5. add isoprprop to precipitate DNA

6. 70% EtOH to wash away salt

7. air dry DNA (not too long)

8. dissolve

steps for magnetic bead method 

1. proteinase K digest and lyse membranes 

2. add silica micro-particles with magnetic cores 

   1. beads slectively absorb DNA

3. Turn on magnetic rod (binds beads with DNA)

   1. alternative magnet : external so beads moved to side and solution can be pipetted off and different one added

4. Wash solution (x2)

   1. turn magnet off to mix beads

5. DNA is eluted from beads and dissolved in next solution

what is chelex method

simple, quick and efficient 

DNA has poor purity (good enough for many optimized PCR tests)

=chelating resin with high affinity for metal ions (Mg)

keeps DNA intact by binding metal ions, preventinf them from being used by harmful enz (ex: nucleases)

usually used for DNA extraction from Guthrie spot or amniotic fluid 

steps for chelex method

1. process sample to isolate cells

   1. guthrie spot : transfer cells from filter paper to solution by incubating spot in microtube with water → spin

   2. amniotic fluid : spin

2. add chelex solution

3. heat sample to 56oC

   1. cell membranes lyse which introduces cellular components into solution

   2. matric in solution absorbs cell lysis products

   3. chelex binds metal ions

   4. DNA is still in nucleus

4. heat to 96oC

   1. nuclear membranes lyse = DNA released

   2. proteins denature

5. spin to collect chelex resin + cellular material 

6. supernatant with DNA → new tube 

   1. make sure no chelex resin leaves with DNA (PCR inhibitor)

   2. result = ssDNA (due to 96oC heating, will not re-anneal to itself)

how is DNA dissolved after extraction

in either sterile DNase free water or TE buffer (recommended for longer storage to prevent degradation)

volume depends on tech or kit protocol 

• V too high = DNA conc too low 

• V too low =  DNA conc too high

DNA must be fully dissolved and homogenous before quantitation 

• if not = inaccurate quantitation 

DNA storage and banking

can be stored at room temp, 4, -20, or -80

• 4oC = DNA in the process of testinf to avoid multiple feeze thaws

• -20oC = short term storage

• -80oC = long term storage of leftover clinical samples

banking = extraction of DNA to store for future testing

• -80 used with a back up at -20

• recommended minimum lenght of storage = 25 years

info on UV spectrophotometry for quantitation

nucleic acid absorbs light maximally at 260nm

proteins at 280nm 

320nm is used as a background reading (proteins and nucleic acids have virtually no absorbance 

DNA conc is directly related to its abs

• 1 optical density unit (OD) = 50ug/mL of dsDNA = 33ug of ssDNA

• use [dsDNS] = abs at 260nm x dilution factor x 50ug/mL

  • dilution factor for DeNovix = 1

• 
  

info on fluorometry for quantitation

fluo dye intercalates in strands of dsDNA

expose to UV → dye becomes excited =fluoresces 

• amount of fluo is direclty realted to conc of dsDNA 

cant use for ssDNA, RNA or protein

need to use DNA standards to calibrate instrument before each time 

create curve to calculate conc. (need to enter dilution factor 

info on capillary electrophoresis for quantitation

small thin tube filled with polymer

seperates DNA by size with electric current

high throughput

software can determine quantity of DNA

conc is calculated by comparing the area of the sample peak to the known concentration of the upper marker

how to determine the acceptibility of DNA sample

purity

• 260/280 ratio

• 260/230 ratio

• scanning graph

quality

• high molecular weight DNA vs degraded

quantity

• is the conc and volume enough for test type

QC of purity 

possible contaminates = protein, phenol, RNA, chaotropic salts and organic material

260/280nm ratio : indicates protein, phenol or RNA contamination 

• acceptable range = 1.7-1.9 

  • <1.7 → protein or phenol 

  • >/= 2 → RNA contamination 

  • ratio close to 1 → no measurable DNA in sample

  • trouble shoot if not in range 

• effect of protein: DNA conc. not overly affected 

• effect of phenol : calculated DNA conc. is higher than real 

2603230nm ratio: indication of organic material contamination

• acceptable range = 1.8-2.2 (at michener → >/=1.8 is ideal)

  • <1.8 → organic material contamination

  • >2.2 → likely not an issue

scanning graph: indication of chemical contamination from chaotropic salt, and if DNA is fully dissolved 

• ideal graph 

  • high, tight peak at 260nm

  • crosses X axis near 300nm

  • big dip in curve at 230nm

  • smooth curve with no jagged portions 

• if 230 dip is shifted to the right → chaotropic salt or phenol

• jagged curve → DNA not fully dissolved 

QC - quality 

determined by amount of HMW DNA present 

methods 

• gel electrophoresis 

  • run DNA on agarose gel to see large band or blob >20kb

  • smear = degraded

• capillary electrophoresis 

  • can display gel like a gel wiht a DNA integrity number (assessment of integrity)