Molecular Diagnostics Nucleic Acid Extraction & Quantitation

Question 1

What can RNA and DNA be used for? (4)

Answer 1

-Genetic testing
-Cancer characterization
-Human identity testing
-Detection and characterization of infectious organisms

Question 2

DNA or RNA:
-Relatively stable
-Long, thin strands
-Avoid excessive/rough pipetting & vortexing

Answer 2

DNA

Question 3

DNA or RNA:
-Small, unstable, easily degraded

Answer 3

RNA

Question 4

When is RNA testing necessary?

Answer 4

-RNA viruses (HIV and HCV)
-Expressed gene detection

Question 5

What are the 3 basic steps when extracting DNA?

Answer 5

-Cell lysis
-Digest proteins
-Purify DNA

Question 6

What type of extraction method is this picture depicting?

Answer 6

Liquid-phase extraction

Question 7

What is the more common method of extraction in the clinical lab?

Answer 7

Solid-phase extraction

Question 8

What are 2 types of solid-phase extraction?

Answer 8

-Silica spun column
-Magnetic bead

Question 9

How is nucleic acid purity measured?

Answer 9

Ultraviolet spectrophotometer

Question 10

Nucleic acids absorb UV light at _____

Answer 10

A260 or 260 nM or OD260

Question 11

Proteins absorb UV light at ______

Answer 11

A280 or 280 nM or OD280

Question 12

What does a purity ratio of <1.6 mean?

Answer 12

Unacceptable for any molecular testing

Question 13

What does a purity ratio between 1.6 and 1.8 mean?

Answer 13

Acceptable for some tests (PCR), but not others (sequencing)

Question 14

What does a purity ratio greater than or equal to 1.8 mean?

Answer 14

Very clean nucleic acid preparation that is good to use for any molecular testing

Question 15

1.0 OD unit at A260 (OD260) = ____ ug/mL dsDNA?

Answer 15

50 ug/mL dsDNA

Question 16

1.0 OD unit at A260 (OD 260) = ____ ug/mL RNA

Answer 16

40 ug/mL RNA

Question 17

50 ug/mL x OD260 reading x dilution factor = ?

Answer 17

dsDNA concentration

Question 18

dsDNA concentration/1000 mL x total sample volume = ?

Answer 18

Yield

Question 19

1 OD260 = 0.498 units
1 OD280 = 0.243 units
What is the ratio?
Acceptable?

Answer 19

2.05
Yes

Question 20

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 = ?

Answer 20

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