Nucleic Acid Quantification

Question 1

After extraction, the concentration and purity of nucleic acids must be assessed.

Common methods include:

Answer 1

Spectrophotometry
Fluorometry

Question 2

– Uses fluorescent dyes that bind specifically to DNA/RNA for more accurate quantification.

Answer 2

Fluorometry

Question 3

– Measures absorbance at 260 nm (DNA/RNA)

Answer 3

Spectrophotometry (Nanodrop Method)

Question 4

Visualizes DNA/RNA integrity and possible degradation.

Answer 4

Agarose Gel Electrophoresis

Question 5

______ appears as a smear on an agarose gel rather than sharp ribosomal RNA bands.

Answer 5

Degraded RNA

Question 6

What is the next step if you already isolated DNA?

Answer 6

Quantity and Quality Checking

Question 7

● What is used for quality checking?

Answer 7

Gel Electrophoresis

Question 8

PCR amplification–_____ng for human genomic DNA

Answer 8

5 to 50 ng

Question 9

DNA and RNA can absorb light at what wavelength?

Answer 9

260 nm

Question 10

● Spectrophotometry is widely used for quantifying DNA;

why????

Answer 10

the primary method for determining the concentration and purity of the sample

Question 11

What is the most crucial step in molbio?

Answer 11

DNA isolation

Provide pure, good quality/ quantity DNA

If not done properly, downstream procedure or experiments will be useless

Question 12

POST DNA ISOLATION

Answer 12

• RNAse treatment if DNA is isolated
• DNAse treatment if RNA is isolated
• Gel Electrophoresis
• Nanodrop Method

Question 13

Why must NA Quatification be done?

Answer 13

Molecular techniques have specific required amounts of sample/ concentration needed

Question 14

Determining Sample Quality
• Ensures that extracted nucleic acids are…

Answer 14

free from contaminants such as proteins, salts, organic solvents, or enzymatic inhibitors.

Question 15

Some experiments require specific DNA/RNA concentrations. Without proper_____\, the experimental conditions may not be optimal.

Answer 15

quantification

Question 16

Poor-quality nucleic acids can lead to….

Answer 16

failed reactions in PCR, sequencing, and other applications.

Question 17

Restriction Enzyme Digestion
• Quantity Required:

Answer 17

0.5 to 10 µg

Question 18

Analyses Using Modifying Enzymes (Ligation & Cloning)
• Quantity Required:

Answer 18

Up to 10 µg

Question 19

Cycle Sequencing
• Quantity Required:

Answer 19

<1 µg

Question 20

PCR Amplification
• Quantity Required:

Answer 20

5 to 50 ng (for human genomic DNA)

Question 21

Amplification-Based SNP Genotyping
• Quantity Required:

Answer 21

10 to 50 ng

Question 22

Array-Based Genotyping
• Quantity Required:

Answer 22

0.5 to 1 µg

Question 23

is a widely used technique for measuring the concentration and purity of nucleic acids (DNA/RNA) by analyzing how they absorb ultraviolet (UV) light.

Answer 23

Spectrophotometry

Question 24

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.

Answer 24

Beer-Lambert’s Law

Question 25

For nucleic acid quantification, spectrophotometers measure absorbance at _______because DNA and RNA absorb light most strongly at this wavelength due to their nitrogenous bases.

Answer 25

260 nm (A260)

Question 26

• __________ 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.

Answer 26

Spectrometer Photometer

Question 27

The_______ generates the light needed for measurement, while the______ analyzes the transmitted or absorbed light and produces a readable output.

Answer 27

spectrometer photometer

Question 28

Parts os spectrophotometry

Answer 28

Light source Collimator (lens) Prism (monochromator) Wavelength selector (Slit) Sample solution (inside the cuvette) Detector (photocell) Digital display

Question 29

Light source

Answer 29

Provides a stable and consistent source of DISPERSED light for absorbance measurement.

Question 30

Lens (Collimator)

Answer 30

Focuses and directs the light beam into a straight path.

Question 31

Monochromator (Prism or Diffraction Grating)

Answer 31

Creates different wavelengths.

Question 32

Wavelength Selector (Slit)

Answer 32

A narrow slit that allows only the desired wavelength to pass through.

Question 33

Sample Solution (in Cuvette)

Answer 33

Holds the nucleic acid sample while allowing UV light to pass through.

Question 34

Detector (Photocell or Photodiode)

Answer 34

Detects the light passing through the sample and converts it into an electrical signal.

Question 35

Digital Display or Meter

Answer 35

Displays the absorbance values for different wavelengths.

Question 36

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.

Answer 36

Fluorometry

Question 37

directly measures fluorescence emitted by dye-bound nucleic acids, making it more accurate and specific, especially for low-concentration samples.

Answer 37

fluorometry

Question 38

Principle of Fluorometry Fluorometry works on the principle of….

Answer 38

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.

Question 39

are highly selective, meaning they bind specifically to DNA, RNA, or proteins.

Answer 39

Fluorescent dyes

Question 40

It is a method which is 1000x more sensitive Why???

Answer 40

Fluorometry Is specifically and selectively binds to DNA/ RNA (will not bind to contaminants)

Question 41

Disadvantages of fluorometry

Answer 41

Expensive Tedious Cannot provide purity ratio

Question 42

Fluorometry The more intense the light…

Answer 42

Higher amounts of DNA

Question 43

Fluorescence will only occur if..

Answer 43

There is binding of dyes + Excitation

Question 44

Components of a Fluorometer

Answer 44

***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

Question 45

determines the average concentration of DNA or RNA in a sample prior to proceeding with downstream experiments.

Answer 45

NA Quantification

Question 46

to know the dilutions needed for every type of an experiment

Answer 46

NA Quantification

Question 47

✔ 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.

Answer 47

Fluorometry

Question 48

Summary of the Fluorometry Process for Nucleic Acid Quantification 1. ______is added to the DNA, RNA, or protein sample. 2. The dye binds specifically to the_____ 3. The sample is exposed to an____ from the fluorometer. 4. The dye emits____ at a longer wavelength. 5. A_____ measures the emitted fluorescence intensity. 6. The nucleic acid concentration is calculated based on a standard curve

Answer 48

Fluorescent dye target molecule excitation beam fluorescence detector

Question 49

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

Answer 49

Spectrophotometry Fluorometry

Question 50

Sensitivity Spectrophotometry Fluorometry

Answer 50

Moderate High

Question 51

Specificity Spectrophotometry Fluorometry

Answer 51

Less specific; affected by contaminants Highly specific

Question 52

Spectrophotometry Advantages

Answer 52

- Simple to use. - Provides purity ratios. - Can analyze a wide range of compounds. - Provides information on contaminants.

Question 53

Fluorometry Advantages

Answer 53

- Highly sensitive, detects very low concentrations. - More specific to target molecules. - Accurate for low-abundance analytes.

Question 54

Spectrophotometry Disadvantages

Answer 54

- Limited sensitivity for low concentrations. - Less selective due to interference from other absorbing species.

Question 55

Fluorometry Disadvantages

Answer 55

- Requires fluorescence labeling or naturally fluorescent compounds. - Longer sample preparation time. - No information on sample purity. - Higher cost due to specialized detectors