Seminar and Lab 03: Absorption Spectrophotometry of Nucleic Acids

Practice flashcards covering cell signaling syllabus, spectrophotometry principles, Lambert-Beer law, and nucleic acid quantification/purity analysis.

Lambert-Beer law

The equation $A = \log(I_0/I) = kcl$ used to calculate absorbance based on light intensity, concentration, and layer thickness.

Absorbance ($A$)

The ability of a substance to absorb radiation, calculated as the logarithm of the ratio of incident light intensity ($I_0$) to transmitted light intensity ($I$).

Specific absorbance coefficient ($k$)

A value characteristic for a given substance and solvent at a specific wavelength, numerically equal to the absorbance of a solution with a concentration of $1\,mol/l$ and layer thickness of $1\,cm$.

Single-beam spectrophotometer

A measuring device where one beam passes first through a standard solution and then through the test sample.

Double-beam spectrophotometer

A device where the radiation beam is split into two; one passes through the standard and the other through the test solution, with the detector indicating the difference in absorbance.

Visible light range

In absorption spectrophotometry, this range covers wavelengths from $\lambda = 390-800\,nm$.

Far Ultraviolet (UV) range

The wavelength range characterized by $\lambda = 10-200\,nm$.

Close Ultraviolet (UV) range

The wavelength range characterized by $\lambda = 200-390\,nm$.

Infrared absorbiometry range

The wavelength range covering $\lambda = 800\,nm-1\,mm$.

Nucleic acid absorption maximum

The wavelength where nucleic acids exhibit peak radiation absorption, specifically at $\lambda = 260\,nm$.

Purine and pyrimidine bases

The components of nucleic acids responsible for radiation absorption due to their conjugated double bonds.

Hypochromic effect

The reduction in light absorption caused by the helical spatial arrangement of polynucleotide strands.

Hyperchromic effect

An increase in the absorption of radiation.

Bathochrome effect

A shift of the absorption band toward longer wavelengths, often associated with an increase in chromophore groups.

Hypsochromic effect

A shift of the absorption band toward lower (shorter) wavelengths.

Chromophoric groups

Groups of atoms responsible for color in the UV/VIS range.

Auxochromic groups

Groups that mainly cause an increase in color intensity and may lead to a bathochromic effect.

dsDNA concentration constant

The assumption that an absorbance of $1$ at $260\,nm$ corresponds to a double-stranded DNA concentration of $50\,\mu g/ml$.

ssDNA concentration constant

The assumption that an absorbance of $1$ at $260\,nm$ corresponds to a single-stranded DNA concentration of $33\,\mu g/ml$.

RNA concentration constant

The assumption that an absorbance of $1$ at $260\,nm$ corresponds to an RNA concentration of $40\,\mu g/ml$.

$A_{260}/A_{280}$ ratio for pure DNA

A purity ratio value of approximately $1.8 - 2.0$ indicating double-stranded DNA free from contaminants.

$A_{260}/A_{280} < 1$ (approx. $0.5$)

A ratio indicating contamination of a DNA sample with proteins.

$$A_{260}/A_{280} > 2$$

A ratio indicating contamination of a DNA sample by RNA, or an RNA sample by DNA.

$A_{260}/A_{230}$ purity range

The typical value range of $2.0 - 2.5$ for pure nucleic acid samples.

Background measurement wavelength

A wavelength of $320\,nm$ or above, used to detect air bubbles, particles, or cuvette smudges as nucleic acids do not absorb here.

Colorimetry

A branch of absorption spectrophotometry covering visible radiation ($390-800\,nm$) used to determine the concentration of colored solutions.

Standard curve (calibration curve)

A graphical representation of the relationship between absorbance and the concentration of a standard substance used for analytical calibration.

Cystic fibrosis presentation requirements

Clinical description, molecular and biochemical basis of the disease, and diagnostics.