DNA Extraction Flashcards
In 1869, the first ________________________ was achieved.
DNA isolation
In 1958, first ___________ was achieved based on ___________ strategies.
DNA extraction; density gradient centrifugation
1950’s: _____________________ was discovered
DNA’s double helix structure
When was DNA polymerase discovered?
1956
What are two early DNA isolation methods?
cesium chloride/ethidium bromide centrifugation and organic “salting out”
How does lysis occur for DNA isolation?
Via salt solution or proteases, or both
DNA purification via columns
+ charged materials bind to = charged DNA, washed with salt solutions to remove unbound material, DNA recovered with water or pH 7-8 buffer to break matrix-DNA bond
Types of PCR-based HLA assays that use genomic DNA
sequence-specific primer (SSP), sequence-specific oglionucleotide probe hybridization (SSOPH), Sanger sequencing-based typing (SBT), quantitative PCR (qPCR), next generation sequencing (NGS), short tandem repeat (STR) analysis
260 nm is the wavelength that _____________. 280 nm is the wavelength that _____________.
DNA absorbs UV light; protein contaminants absorb UV light.
Acceptable A260/A280 ratio for purified DNA
between 1.8 and 2.0
Protocol: Salting Out Method
- Disruption and lysis of cell membrane using a buffer containing a detergent (eg. SDS) and proteinase K at 37 C.
- Unwanted components like proteins are precipitated out using high salt concentrations.
- After centrifugation, supernatant containing DNA is removed by pipetting into a separate tube.
- Ethanol is added to supernatant and DNA precipitates out as visible white strands.
- Following centrifugation to pellet the DNA, ethanol is removed and DNA pellet air dried.
- Dried pellet is dissolved in Tris-EDTA (TE) or reagent grade water.
Protocol: Magnetic Bead Extraction
- Samples are lysed using a buffer containing a detergent and proteinase K.
- After cell lysis, magnetic particles are used to selectively purify DNA molecules from the sample lysates.
- The beads are isolated with a magnet and the liquid removed.
- The bead/DNA complex is washed with an ethanol buffer solution.
- DNA is eluted off the magnetic beads using molecular biology grade water.
If DNA is stored at -20 C or -80 C, what elution reagent is preferred?
Tris-EDTA
DNase-free water can be used as an eluate, but DNA stored in H2O is subject to what?
acid hydrolysis
What is Tris-EDTA?
A buffer solution that helps maintain the stability of nucleic acids during sample preparation, electrophoresis, and enzymatic reactions; used for DNA sequencing and PCR applications.
What is elution?
A process that uses a solvent to extract one material from other; process of extracting a substance that is adsorbed to another by washing it with a solvent.
How can carry-over contamination be avoided during DNA extraction?
physical separation between the pre-amplification and post-amplification areas; use of DNase-free consumables, aerosol barrier pipette tips, laminar-flow hood, dedicated sets of pipettes for extraction and PCR set-up
Daily cleaning of all work surfaces should be performed with ____________. Weekly cleaning should be performed with _____________.
70% ethanol; 10% bleach followed by 70% ethanol
What is an easy way to detect contamination in the lab?
PCR amplification of a wipe of work surfaces to detect contamination of human genomic and amplified DNA.
What can you do if your DNA concentration from a column extraction is too low?
- Concentrate DNA by alcohol precipitation by adding a volume of cold ethanol that is twice the volume of the suspension. Vortex and centrifuge on highest speed. Pipette out as much alcohol as possible and add water to sample. Vortex and repeat quantitation and purity check.
- Extract an additional sample with increased volume, or, if WB was used, prepare a buffy coat.
- Could be indicative of inefficient cell lysis or protein degradation of sample due to insufficient mixing/incubation time. Repeat purification procedure. Make sure sample is thoroughly digested.
- DNA concentration can be increased by decreasing the volume of elution buffer.
What does a high A260/A280 ratio mean?
DNA sample may be contaminated with RNA, which won’t interfere with PCR but could interfere in downstream applications.
What does a low A260/A280 ratio mean?
Protein contamination; if ratio is lower than 1.75, PCR will probably be affected
Reason for low/poor quality PCR product and/or PCR reaction failure but A260/A280 was within range
Most frequent cause is alcohol contamination.
