MIDTERM LEC: MEASUREMENT OF NUCLEIC ACID QUALITY & QUANTITY Flashcards

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1
Q

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

A

ELECTROPHORESIS
SPECTROPHOTOMETRY
FLUOROMETRY
MICROFLUIDICS

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2
Q

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

A

ELECTROPHORESIS

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3
Q

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

A

SPECTROPHOTOMETRY

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4
Q

Measurement of emitted fluorescent light

A

FLUOROMETRY

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5
Q

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

A

MICROFLUIDICS

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6
Q

▪ 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

A

ELECTROPHORESIS

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7
Q

Fluorescent dyes used in ELECTROPHORESIS

A

ethidium bromide, SybrGreen I & II, silver stain

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8
Q

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

A

ELECTROPHORESIS

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9
Q

Nucleic acids absorb light at 260 nm through the adenine residues

A

SPECTROPHOTOMETRY

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10
Q

Beer-Lambert Law:

A

A = ∈bc

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11
Q

A = ∈bc

A

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

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12
Q

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

A

directly

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13
Q

molar absorptivity for DNA???

A

50 mg/L or ug/mL

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14
Q

molar absorptivity for RNA???

A

40 mg/L or ug/mL

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15
Q

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

A

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

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16
Q

SPECTROPHOTOMETRY

determine concentration:

A

spectrophotometer reading in absorbance units x appropriate conversion factor

17
Q

SPECTROPHOTOMETRY

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

A

Absorbance reading x conversion factor x dilution factor

18
Q

ESTIMATION OF PURITY OF NUCLEIC ACID
Detection of contaminants: _____

A

reading the concentration over a range of wavelength

19
Q

ESTIMATION OF PURITY OF NUCLEIC ACID

Indication of contamination: _____

A

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

20
Q

A260/A280 DNA= ______

A

1.6 to 2.00 times more than the absorbance at 280 nm

21
Q

A260/A280 RNA=_____

A

2.0 to 2.3

22
Q

A260/A280 = <1.6

A

contaminated

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

23
Q

▪ 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

A

UV Spectrometry

24
Q

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

A

NanoDrop Spectrophotometry

25
Q

● 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

A

FLUOROMETRY (FLUORESCENT SPECTROSCOPY)

26
Q

Other DNA-specific dyes in FLUOROMETRY (FLUORESCENT SPECTROSCOPY)

A

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

27
Q

stain used for RNA IN FLUOROMETRY (FLUORESCENT SPECTROSCOPY)

A

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

28
Q

▪ 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

A

ABSORPTION & FLUOROMETRY READINGS MAY NOT ALWAYS AGREE

29
Q

▪ 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

A

MICROFLUIDICS

30
Q

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

A

RNA integrity number