Nucleic Acid Quantification Flashcards
After extraction, the concentration and purity of nucleic acids must be assessed.
Common methods include:
Spectrophotometry
Fluorometry
– Uses fluorescent dyes that bind specifically to DNA/RNA for more accurate quantification.
Fluorometry
– Measures absorbance at 260 nm (DNA/RNA)
Spectrophotometry (Nanodrop Method)
Visualizes DNA/RNA integrity and possible degradation.
Agarose Gel Electrophoresis
______ appears as a smear on an agarose gel rather than sharp ribosomal RNA bands.
Degraded RNA
What is the next step if you already isolated DNA?
Quantity and Quality Checking
● What is used for quality checking?
Gel Electrophoresis
PCR amplification–_____ng for human genomic DNA
5 to 50 ng
DNA and RNA can absorb light at what wavelength?
260 nm
● Spectrophotometry is widely used for quantifying DNA;
why????
the primary method for determining the concentration and purity of the sample
What is the most crucial step in molbio?
DNA isolation
Provide pure, good quality/ quantity DNA
If not done properly, downstream procedure or experiments will be useless
POST DNA ISOLATION
• RNAse treatment if DNA is isolated
• DNAse treatment if RNA is isolated
• Gel Electrophoresis
• Nanodrop Method
Why must NA Quatification be done?
Molecular techniques have specific required amounts of sample/ concentration needed
Determining Sample Quality
• Ensures that extracted nucleic acids are…
free from contaminants such as proteins, salts, organic solvents, or enzymatic inhibitors.
Some experiments require specific DNA/RNA concentrations. Without proper_____\, the experimental conditions may not be optimal.
quantification
Poor-quality nucleic acids can lead to….
failed reactions in PCR, sequencing, and other applications.
Restriction Enzyme Digestion
• Quantity Required:
0.5 to 10 µg
Analyses Using Modifying Enzymes (Ligation & Cloning)
• Quantity Required:
Up to 10 µg
Cycle Sequencing
• Quantity Required:
<1 µg
PCR Amplification
• Quantity Required:
5 to 50 ng (for human genomic DNA)
Amplification-Based SNP Genotyping
• Quantity Required:
10 to 50 ng
Array-Based Genotyping
• Quantity Required:
0.5 to 1 µg
is a widely used technique for measuring the concentration and purity of nucleic acids (DNA/RNA) by analyzing how they absorb ultraviolet (UV) light.
Spectrophotometry
Spectrophotometry
The principle is based on______, which states that the absorbance of light by a solution is proportional to the concentration of the absorbing molecules.
Beer-Lambert’s Law
For nucleic acid quantification, spectrophotometers measure absorbance at _______because DNA and RNA absorb light most strongly at this wavelength due to their nitrogenous bases.
260 nm (A260)
• __________ Measures the intensity of light at specific wavelengths.
• __________ Detects the amount of light that passes through the sample and converts it into an electrical signal.
Spectrometer
Photometer
The_______ generates the light needed for measurement, while the______ analyzes the transmitted or absorbed light and produces a readable output.
spectrometer
photometer
Parts os spectrophotometry
Light source
Collimator (lens)
Prism (monochromator)
Wavelength selector (Slit)
Sample solution (inside the cuvette)
Detector (photocell)
Digital display
Light source
Provides a stable and consistent source of DISPERSED light for absorbance measurement.
Lens (Collimator)
Focuses and directs the light beam into a straight path.
Monochromator (Prism or Diffraction Grating)
Creates different wavelengths.
Wavelength Selector (Slit)
A narrow slit that allows only the desired wavelength to pass through.
Sample Solution (in Cuvette)
Holds the nucleic acid sample while allowing UV light to pass through.
Detector (Photocell or Photodiode)
Detects the light passing through the sample and converts it into an electrical signal.
Digital Display or Meter
Displays the absorbance values for different wavelengths.
is a highly sensitive method used to quantify nucleic acids (DNA/RNA) and proteins by utilizing fluorescent dyes that specifically bind to the target molecules.
Fluorometry
directly measures fluorescence emitted by dye-bound nucleic acids, making it more accurate and specific, especially for low-concentration samples.
fluorometry
Principle of Fluorometry
Fluorometry works on the principle of….
fluorescence, where a
fluorescent dye that is bound to DNA absorbs light at a specific wavelength (excitation) and then emits light at a longer wavelength (emission).
This emitted light is directly proportional to the amount of nucleic acid or protein in the sample.
are highly selective, meaning they bind specifically to DNA, RNA, or proteins.
Fluorescent dyes
It is a method which is 1000x more sensitive
Why???
Fluorometry
Is specifically and selectively binds to DNA/ RNA (will not bind to contaminants)
Disadvantages of fluorometry
Expensive
Tedious
Cannot provide purity ratio
Fluorometry
The more intense the light…
Higher amounts of DNA
Fluorescence will only occur if..
There is binding of dyes + Excitation
Components of a Fluorometer
Emitter (light source)
Excitation beam
Sample with Fluorescent Dye in Cuvette or Microplate
Emitted Beam
Detector (Photomultiplier Tube or Photodiode)
Digital Display or Computer Interface
determines the average
concentration of DNA or RNA in a
sample prior to proceeding with
downstream experiments.
NA Quantification
to know the dilutions needed for
every type of an experiment
NA Quantification
✔ Higher Sensitivity: Detects DNA/RNA in picogram (pg) levels.
✔ Higher Specificity: Selective dyes reduce interference from contaminants.
✔ More Accurate for Low-
Concentration Samples: Unlike spectrophotometry, it does not overestimate due to contaminants like proteins or salts.
Fluorometry
Summary of the Fluorometry Process for Nucleic Acid Quantification
- ______is added to the DNA, RNA, or protein sample.
- The dye binds specifically to the_____
- The sample is exposed to an____ from the fluorometer.
- The dye emits____ at a longer wavelength.
- A_____ measures the emitted fluorescence intensity.
- The nucleic acid concentration is calculated based on a standard curve
Fluorescent dye
target molecule
excitation beam
fluorescence
detector
Principle
Measures absorbance of light by a sample at specific wavelengths based on Beer-Lambert’s Law.
Measures fluorescence emitted by a sample after excitation at a specific wavelength
Spectrophotometry
Fluorometry
Sensitivity
Spectrophotometry
Fluorometry
Moderate
High
Specificity
Spectrophotometry
Fluorometry
Less specific; affected by contaminants
Highly specific
Spectrophotometry
Advantages
- Simple to use.
- Provides purity ratios.
- Can analyze a wide range of compounds.
- Provides information on contaminants.
Fluorometry
Advantages
- Highly sensitive, detects very low
concentrations. - More specific to target molecules.
- Accurate for low-abundance analytes.
Spectrophotometry
Disadvantages
- Limited sensitivity for low concentrations.
- Less selective due to interference from
other absorbing species.
Fluorometry
Disadvantages
- Requires fluorescence labeling or naturally
fluorescent compounds. - Longer sample preparation time.
- No information on sample purity.
- Higher cost due to specialized detectors
