360 - Molecular Techniques Flashcards
The target n________ _____d for detection will determine the starting material for extraction.
nucleic acid
The selection of starting material is dependent on this
the target nucleic acid
potential target nucleic acids
chromosomal DNA, mitochondrial DNA, circulating fetal DNA, and microbial DNA & RNA
The three basic steps in DNA extraction and purification:
cell disruption, separation of DNA from cellular material, and concentration of the DNA
how are DNA cell lysates produced?
DNA cell lysates are produced by disrupting cell walls and membranes
Lysis solutions usually contain one or more of the following reagents:
chaotropic salts – guanidinium isothiocyanate, urea, and sodium dodecyl sulphate (SDS); detergents – SDS; and an alkaline denaturant – sodium hydroxide
NOTE: these agents denature proteins resulting in the disruption of cell membranes and the release of the cellular contents, including DNA
porteinase K can be added
This is also added to lysis buffers to chelate metal ions which are required as cofactors for DNases
EDTA
This can be added to extraction to remove residual RNA
RNase
Processing before lysis: tissue biopsies
They can be disrupted by homogenization using stainless steel beads in a bead mixer or by grinding liquid nitrogen frozen tissue with a mortar or pestle
T or F. Nucleic acids can be extracted from formalin fixed paraffin embedded tissue
T!
Gram-positive bacteria require enzymatic digestion with THIS before cell lysis
lysozyme
Fungal cell walls can be disrupted by enzymes or by using c______ t_________ b_____ in a bead mixer
carbon tungsten beads
how can cell lysates be further purified to separate DNA from other cellular components?
can be cleared by liquid extraction/purification methods like phenol/chloroform or by solid phase isolation like magnetic silica beads
- liquid extraction/purification methods = cellular components are precipitated by centrifugation or removed by filtration
- magnetic bead extraction/purification method = binding of the DNA to the bead surface by adsorptive or ionic interactions
Qiagen Symphony
an automated nucleic acid purification platform
how does the Qiagen Symphony work?
- samples incubated in a lysis buffer containing a chaotropic salt, and usually, Proteinase K
- cells lyse and nucleic acids + other cellular components released
- magnetic silica beads added to lysate, and nucleic acids adsorb to the silica
- magnetic rod transfers the bound nucleic acid material to a series of reaction vessels where the DNA is washed to remove contaminants
- DNA eluted in a low salt buffer.
DNA and nucleotides absorb at which wavelength
260 nm
how do we calculate nucleic acids in a pure solution?
50 μg/mL of double-stranded DNA has an A260 of 1
33 μg/mL of single-stranded DNA has an A260 of 1
40 μg/mL of single-stranded RNA has an A260 of 1
T or F. Both nucleic acid and proteins absorb UV light at 260 and 280 nm.
T!
how can protein contamination of nucleic acid solutions be assessed?
by analyzing the ratio of absorbance at 260:280 nm
pure dsDNA has an A260/A280 ratio of 1.7 to 1.9
pure single-stranded RNA has an A260/A280 ratio of approximately 2.0
T or F. The A260/A280 ratio is affected by alkaline pH
F! acidic pH; slightly alkaline buffer is the preferred solvent
These contaminants can be detected by analyzing the absorbance at 230 nm
carbohydrates, phenol and guanidine
The A260/A230 ratio of pure nucleic acid preparation will be greater than the A260/A280 ratio
what is a Nanodrop?
assess nucleic acid concentration and purity but
used instead of a spec (Nanodrop is faster & more convenient!)
how does a Nanodrop work?
- small volumes of the sample (1-2 μL) are pipetted onto the end of a fibre optic cable
- a second cable is placed on the opposite side of the drop of sample to form a small gap
- a pulsed xenon flash lamp provides the light source and a spectrometer utilizing a linear charged couple detect array is used to analyze the light after passing through the sample
- a computer algorithm calculates the concentration of nucleic acid
endpoint or conventional PCR
any PCR protocol which uses agarose gel electrophoresis to visualize the PCR amplicons