Molecular Diagnostics Nucleic Acid Extraction & Quantitation Flashcards
What can RNA and DNA be used for? (4)
-Genetic testing
-Cancer characterization
-Human identity testing
-Detection and characterization of infectious organisms
DNA or RNA:
-Relatively stable
-Long, thin strands
-Avoid excessive/rough pipetting & vortexing
DNA
DNA or RNA:
-Small, unstable, easily degraded
RNA
When is RNA testing necessary?
-RNA viruses (HIV and HCV)
-Expressed gene detection
What are the 3 basic steps when extracting DNA?
-Cell lysis
-Digest proteins
-Purify DNA
What type of extraction method is this picture depicting?
Liquid-phase extraction
What is the more common method of extraction in the clinical lab?
Solid-phase extraction
What are 2 types of solid-phase extraction?
-Silica spun column
-Magnetic bead
How is nucleic acid purity measured?
Ultraviolet spectrophotometer
Nucleic acids absorb UV light at _____
A260 or 260 nM or OD260
Proteins absorb UV light at ______
A280 or 280 nM or OD280
What does a purity ratio of <1.6 mean?
Unacceptable for any molecular testing
What does a purity ratio between 1.6 and 1.8 mean?
Acceptable for some tests (PCR), but not others (sequencing)
What does a purity ratio greater than or equal to 1.8 mean?
Very clean nucleic acid preparation that is good to use for any molecular testing
1.0 OD unit at A260 (OD260) = ____ ug/mL dsDNA?
50 ug/mL dsDNA
1.0 OD unit at A260 (OD 260) = ____ ug/mL RNA
40 ug/mL RNA
50 ug/mL x OD260 reading x dilution factor = ?
dsDNA concentration
dsDNA concentration/1000 mL x total sample volume = ?
Yield
1 OD260 = 0.498 units
1 OD280 = 0.243 units
What is the ratio?
Acceptable?
2.05
Yes
A DNA extraction resulted in 150 uL total volume. The sample was diluted 1:4 and gave an OD260 reading of 0.436 units.
dsDNA conc. = ?
Yield = ?
dsDNA = 50 ug/mL x 0.436 units x 4 = 87.2 ug/mL
Yield = 87.2/1000 x 150 uL = 13.08 ug DNA
