DNA EXTRACTION Chloe Swinfield

Why Extract DNA? Purified DNA ready for direct use in downstream applications Removes proteins, nucleases and other impurities and any potential PCR inhibitors (EDTA, FFPE, clothing dyes) More stable/less degradation/easier and cheaper to store

Many Extraction Techniques Spin Columns Manual Qiacube Magnetic beads Qiasymphony Liquid/Liquid Phenol/Chloroform FlexiGene

Many Extraction Techniques Sample Quality Sample Number Cost Time Simplicity and Experience Automation Safety

Qiagen spin columns Qiagen Mini Kit The QIAamp DNA Mini Kit provides silica- membrane-based nucleic acid purification from tissues, swabs, CSF, blood, body fluids, or washed cells from urine. Reduces hands-on preparation time to 20 minutes. Purification of DNA using the QIAamp DNA Mini Kit can be automated on the QIAcube.

Lysis Break down cells to access DNA in the nucleus Lysis buffer Chaotropic salts Destabilise hydrogen bonds, hydrophobic interactions, proteins (including nucleases) Disrupts the association of nucleic acids with water in preparation to bind to silica membrane Detergent (protein lysis and solubisation) Enzyme Heat

Purification - Bind Separate purified DNA from cell material and any PCR inhibitors Ethanol is added to enhance the binding of DNA to silica Load sample to column Centrifuge DNA binds to the membrane and remaining lysate discarded

Purification - Wash Impurities and proteins should now have passed through the column and been discarded The membrane, however, is still dirty with residual proteins and salt, if using blood it may be tinted yellow or brown The column is washed with buffers to remove any residual impurities There are typically 2 washes with a centrifuge step after each Wash 1 will contain a low amount of chaotropic salt to remove any remaining proteins and coloured contaminants Wash 2 contains a high concentration of ethanol to remove the remaining salts. Salts MUST be removed for good DNA yields and purity. Wash 2 can be repeated to ensure this. All ethanol MUST be removed so that the DNA can be successfully removed/eluted from the silica membrane. Centrifuge the sample until the column is completely dry Effect of Residual Salts The 3730 DNA Analyzer is especially susceptible to salt in samples from template preparation. The negative ions in salts can be preferentially injected into the capillary array during electrokinetic injection, leading to lower signal. In addition, the negative ions compete and interfere with the injection of larger DNA extension fragments, leading to shortened read lengths. If salts and unincorporated dyes are not removed from the sequencing reaction, they will compete with extension fragments during electrokinetic injection and result in weak signals

Elute When elution buffer is added the nucleic acids become hydrated and will release from the membrane. If the column still has ethanol on it, the nucleic acids will not fully rehydrate Buffer AE in the QIAamp mini kit. DNA more stable at a slightly basic pH and will dissolve more easily in a basic buffer. Elution of DNA can be maximised by allowing the buffer to sit in the membrane for a few minutes before centrifugation. Can be repeated

Qiacube Automated processing of the Qiagen spin columns Capacity: up to 12 samples per run Optical & Ultrasonic sensors: check samples, consumables and liquid loading for each protocol run

Qiacube The QIAcube is preinstalled with protocols for purification of plasmid DNA, genomic DNA, RNA, viral nucleic acids, and proteins. Reduces hands on sample prep Reduces human error and human introduced contamination Less control, cannot stop the process once started to observe/optimise. Clogging of the spin column

Qiasymphony

Qiasymphony Silica magnetic bead rather than spin column Larger surface area for DNA to bind to Removes spin column clogging problem

Qiasymphony Samples loaded in batches of 24 4 batches of 24 can be loaded and queued on the machine at one time Sample carrier can be removed once sample has been taken for lysis and replaced with another First run of 24 takes around 1.5hrs 1 hour for subsequent runs Samples can be queued and run overnight

Qiasymphony All plastic wear (rod covers and sample prep cartridges) pipette tips and reagents are loaded and scanned before a run If there is an insufficient amount for the number of samples you would like to process an error message will display There are four elution rack holders (Qiagen elution tubes or 2D barcoded tubes). One is cooled for overnight runs or RNA/more sensitive work

Qiasymphony Advantages More samples than Qiacube or manual extraction Magnetic beads more efficient and eliminate clogging Sealed reagent cartridge and plasticwear reduce human handling UV light Tip protectors/drip catchers Simple batch set up on screen

Qiasymphony Disadvantages Expensive (catalogue price £546 for 144 preps) High sample volume required (420µl buffy coat midi kit) Quite limited in elution volumes/all protocols must be prepared by Qiagen As previously mentioned, automation limits the amount of alterations and optimisations you may want to do when samples require more work

Phenol/Chloroform Phenol Chloroform (~1:1) is added to your aqueous sample containing the required DNA as well as proteins Phenol and water are immiscible so two phases form. Phenol is more dense (chloroform increases the density of the phenol) and so the organic phase sinks to the bottom The two phases are mixed causing proteins to denature and partition into the less polar organic phenol phase. The sample is centrifuged Polar DNA remains in the polar aqueous phase whilst proteins remain in the less polar organic phase. The aqueous layer can then be transferred and the organic phase discarded The DNA in the solution can now be precipitated, washed and resuspended

FlexiGene Lyse cells and spin down to form a pellet (discard supernatant) Add denaturation buffer to denature proteins and incubate Add isopropanol to precipitate DNA. Sample must be mixed thoroughly by inversion. Centrifuge to form pellet Isopropanol removed and sample washed with ethanol Sample is centrifuged, the ethanol supernatant removed and the pellet air dried. Required storage buffer is then added to the pellet and the sample incubated at 65⁰C until the pellet dissolves DNA less soluble in Isopropanol than water so it will fall out of solution faster and at a lower concentration. However so will any salts. So wash with ethanol to remove any salts.

Advantages and Disadvantages Cheap (Flexigene £215 for 250 extractions) More control throughout the process, buffers, incubation times Therefore good for poorer quality samples/mixed standard of samples Good yield Long Labour intensive Removing supernatants/resuspending pellets can be fiddly and requires some experience of the technique