MIDTERM LEC: MEASUREMENT OF NUCLEIC ACID QUALITY & QUANTITY

Question 1

WHAT ARE THE 4 METHODS MEASUREMENT OF THE QUALITY & QUANTITY OF DNA & RNA???

Answer 1

ELECTROPHORESIS
SPECTROPHOTOMETRY
FLUOROMETRY
MICROFLUIDICS

Question 2

Separation of
particles through a solution or
matrix under the force of an
electric current

Answer 2

ELECTROPHORESIS

Question 3

Technique that uses light absorption to measure the concentration of an analyte in solution

Answer 3

SPECTROPHOTOMETRY

Question 4

Measurement of emitted fluorescent light

Answer 4

FLUOROMETRY

Question 5

Science & technology of systems that process or manipulate small
amounts of fluid, using channels measuring from tens to hundreds of micrometers

Answer 5

MICROFLUIDICS

Question 6

▪ DNA & RNA can be analyzed for quality (detection & size analysis) by resolving an aliquot of the isolated sample on an agarose gel
▪ Uses an electric current to propel charged biomolecules through a porous gel matrix at a rate that is the function of the charge, size, & shape of the molecules

Answer 6

ELECTROPHORESIS

Question 7

Fluorescent dyes used in ELECTROPHORESIS

Answer 7

ethidium bromide, SybrGreen I & II, silver stain

Question 8

Appearance of DNA on agarose gels depends on the type of DNA isolated

Answer 8

ELECTROPHORESIS

Question 9

Nucleic acids absorb light at 260 nm through the adenine residues

Answer 9

SPECTROPHOTOMETRY

Question 10

Beer-Lambert Law:

Answer 10

A = ∈bc

Question 11

A = ∈bc

Answer 11

❖ A = absorbance
❖ ∈ = molar absorptivity (L/mol-cm), 50 for dsDNA, 40 for RNA
❖ b = path length (cm)
❖ c = concentration (mg/L)

Question 12

Absorbance is _____ proportional to the concentration of the nucleic acid in the sample

Answer 12

directly

Question 13

molar absorptivity for DNA???

Answer 13

50 mg/L or ug/mL

Question 14

molar absorptivity for RNA???

Answer 14

40 mg/L or ug/mL

Question 15

Using absorptivity, as a conversion factor from optical density to concentration:

Answer 15

At 260 nm, 1 optical density unit (or absorbance unit) = 50 mg/L (or 50 μg/mL) of dsDNA & 40 μg/mL of RNA

Question 16

SPECTROPHOTOMETRY

determine concentration:

Answer 16

spectrophotometer reading in absorbance units x appropriate conversion factor

Question 17

SPECTROPHOTOMETRY

If the DNA/RNA preparation require dilution before spectrophotometry, to determine concentration:

Answer 17

Absorbance reading x conversion factor x dilution factor

Question 18

ESTIMATION OF PURITY OF NUCLEIC ACID
Detection of contaminants: _____

Answer 18

reading the concentration over a range of wavelength

Question 19

ESTIMATION OF PURITY OF NUCLEIC ACID

Indication of contamination: _____

Answer 19

absorbance over background at any wavelength other than the A260 maxima of the nucleic acid

Question 20

A260/A280 DNA= ______

Answer 20

1.6 to 2.00 times more than the absorbance at 280 nm

Question 21

A260/A280 RNA=_____

Answer 21

2.0 to 2.3

Question 22

A260/A280 = <1.6

Answer 22

contaminated

Most likely contaminant: protein (absorbs light at 280 nm through the aromatic tryptophan & tyrosine residues)

Question 23

▪ Standard nucleic acid quantitation ▪ Nucleic acid sample is placed into quartz cuvette, which is then placed inside the UV spectrometer
▪ UV light passed through the sample at a specified path length, & the absorbance of the sample at specific wavelengths is measured
▪ Does not require additional reagents/incubation time

Answer 23

UV Spectrometry

Question 24

Similar in principle with the previous, but has many additional capabilities ▪ Functions by combining fiber optic technology & natural surface tension properties
▪ Accompanied by special software to enable analysis of signal from small quantities of sample
▪ Displays the entire absorbance spectrum of the sample in graphical form 🡪 allows detection of contaminants
▪ Capable of determining a wide range of sample concentrations w/o requiring serial dilutions

Answer 24

NanoDrop Spectrophotometry

Question 25

● Measures fluorescence related to DNA concentration in association with DNA-specific fluorescent dyes ● Early methods: 3,5-diaminobenzoic acid �HCl (DABA), combined with alpha-methylene aldehydes (deoxyribose) to yield a fluorescent product ● Modern methods: DNA-specific dye Hoechst 33258, combined with adenine-thymine base pairs in the minor groove of the DNA double helix ⮚ Fluorometric determination of DNA concentration: down to 200 ng DNA/mL

Answer 25

FLUOROMETRY (FLUORESCENT SPECTROSCOPY)

Question 26

Other DNA-specific dyes in FLUOROMETRY (FLUORESCENT SPECTROSCOPY)

Answer 26

PicoGreen = detection down to 25 pg/mL concentrations OliGreen = detection down to 100 pg/mL of ssDNA

Question 27

stain used for RNA IN FLUOROMETRY (FLUORESCENT SPECTROSCOPY)

Answer 27

SybrGreen II RNA gel stain is used (sensitivity = 2 ng/mL)

Question 28

▪ These methods recognize different targets ❖ Single nucleotides do not bind to fluorescent dyes, but they can absorb ultraviolet light ▪ Absorption measurements do not distinguish between DNA & RNA ▪ Deciding which instrument to use is at the discretion of the laboratory: ❖ Most use spectrophotometry because the samples can be read directly without staining or mixing with dye ❖ Methods requiring accurate measurements of low amounts of DNA/RNA (in the range of 10 to 100 ng/mL), fluorometry may be preferred

Answer 28

ABSORPTION & FLUOROMETRY READINGS MAY NOT ALWAYS AGREE

Question 29

▪ Sample is applied to a multi-well chip & then moves through microchannels across a detector ▪ Instrument software generates images in electropherogram (peak) or gel (band) configurations ▪ RNA integrity number: quantification estimate for RNA, determined as a standard measure of RNA integrity ▪ Uses a minimal volume of sample (as low as 1 μL) & can test multiple samples simultaneously ▪ Useful for analysis of studies on small RNAs (microRNAs) in eukaryotes & gene expression in bacteria

Answer 29

MICROFLUIDICS

Question 30

quantification estimate for RNA, determined as a standard measure of RNA integrity

Answer 30

RNA integrity number