Spectrophotometer for Nucleic Acid Measurements
Encyclopedia
A Spectrophotometer for Nucleic Acid Measurement is used in
molecular biology
. Analysis of nucleic acid
s is commonly performed to determine the average concentrations of DNA
or RNA
present in a mixture, as well as their purity. To date, there are two main approaches used by scientists to quantitate DNA or RNA. These are spectrophotometric analysis
and fluorescence tagging
.
light in a specific pattern. In the case of DNA and RNA, a sample that is exposed to ultraviolet light at a wavelength
of 260 nanometre
s (nm) will absorb that ultraviolet light. The resulting effect is that less light will strike the photodetector
and this will produce a higher optical density (OD)
Optical Density= Log (Intensity of Incident Light / Intensity of
Transmitted Light)
In practical terms, a sample that contains no DNA or RNA should not
absorb any of the ultraviolet light and therefore produce an OD of 0
Optical Density=Log (100/100)=0
When using spectrophotometric analysis to determine the concentration of DNA or RNA, the Beer-Lambert law
is used to determine unknown concentrations without the need for standard curves. In essence, the Beer Lambert Law makes it possible to relate the amount of light absorbed to the concentration of the absorbing molecule. The following absorbance units to nucleic acid concentration conversion factors are used to convert OD to concentration of unknown nucleic acid samples:
A260 dsDNA = 50 µg/ml
A260 ssDNA = 37 µg/ml
A260 ssRNA = 40 µg/ml
, simply multiply the OD by the conversion factor
to determine the concentration.
Example, a 2.0 OD dsDNA sample corresponds to a sample with a 100 ug/ml concentration.
When using a path length that is shorter than 10mm, the resultant OD will be reduced by a factor of 10/path length. Using the example above with a 3 mm path length, the OD for the 100 ug/ml sample would be reduced to 0.6. To normalize the concentration to a 10mm equivalent, the following is done:
0.6 OD X (10/3) * 50 ug/ml=100 ug/ml
Most spectrophotometers, like the AQ-07 Nucleic Acid Meter, allow selection of the nucleic acid type and path length such that resultant concentration is normalized to the 10mm path length which is based on the principles of Beer's law.
1 A260 unit dsDNA = 50 µg
1 A260 unit ssDNA = 37 µg
1 A260 unit ssRNA = 40 µg
methods with dye
s that are highly specific to DNA or RNA. The benefit of fluorescence quantitation of DNA and RNA is the improved sensitivity over spectrophotometric analysis
. Although, that increase in sensitivity comes at the cost of a higher price per sample and a lengthier sample preparation process. Additionally, there is not currently a fluorescence method to determine protein contamination of a DNA sample that is similar to the 260 nm/280 nm spectrophotometric version.
molecular biology
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
. Analysis of nucleic acid
Nucleic acid
Nucleic acids are biological molecules essential for life, and include DNA and RNA . Together with proteins, nucleic acids make up the most important macromolecules; each is found in abundance in all living things, where they function in encoding, transmitting and expressing genetic information...
s is commonly performed to determine the average concentrations of DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
or RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
present in a mixture, as well as their purity. To date, there are two main approaches used by scientists to quantitate DNA or RNA. These are spectrophotometric analysis
Spectrophotometry
In chemistry, spectrophotometry is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength...
and fluorescence tagging
Fluorescent tag
In molecular biology and biotechnology, a fluorescent tag is a part of a molecule that researchers have attached chemically to aid in detection of the molecule to which it has been attached. The tag is some kind of fluorescent molecule...
.
Spectrophotometric analysis
One of the more commonly used practices to quantitate DNA or RNA is the use of spectrophotometric analysis using a spectrophotometer like the AQ-07. Spectrophotometric analysis is based on the principles that nucleic acids absorb ultravioletUltraviolet
Ultraviolet light is electromagnetic radiation with a wavelength shorter than that of visible light, but longer than X-rays, in the range 10 nm to 400 nm, and energies from 3 eV to 124 eV...
light in a specific pattern. In the case of DNA and RNA, a sample that is exposed to ultraviolet light at a wavelength
Wavelength
In physics, the wavelength of a sinusoidal wave is the spatial period of the wave—the distance over which the wave's shape repeats.It is usually determined by considering the distance between consecutive corresponding points of the same phase, such as crests, troughs, or zero crossings, and is a...
of 260 nanometre
Nanometre
A nanometre is a unit of length in the metric system, equal to one billionth of a metre. The name combines the SI prefix nano- with the parent unit name metre .The nanometre is often used to express dimensions on the atomic scale: the diameter...
s (nm) will absorb that ultraviolet light. The resulting effect is that less light will strike the photodetector
Photodetector
Photosensors or photodetectors are sensors of light or other electromagnetic energy. There are several varieties:*Active pixel sensors are image sensors consisting of an integrated circuit that contains an array of pixel sensors, each pixel containing a both a light sensor and an active amplifier...
and this will produce a higher optical density (OD)
Calculations
The Optical Density is generated from equation:Optical Density= Log (Intensity of Incident Light / Intensity of
Transmitted Light)
In practical terms, a sample that contains no DNA or RNA should not
absorb any of the ultraviolet light and therefore produce an OD of 0
Optical Density=Log (100/100)=0
When using spectrophotometric analysis to determine the concentration of DNA or RNA, the Beer-Lambert law
Beer-Lambert law
In optics, the Beer–Lambert law, also known as Beer's law or the Lambert–Beer law or the Beer–Lambert–Bouguer law relates the absorption of light to the properties of the material through which the light is travelling.-Equations:The law states that there is a logarithmic dependence between the...
is used to determine unknown concentrations without the need for standard curves. In essence, the Beer Lambert Law makes it possible to relate the amount of light absorbed to the concentration of the absorbing molecule. The following absorbance units to nucleic acid concentration conversion factors are used to convert OD to concentration of unknown nucleic acid samples:
A260 dsDNA = 50 µg/ml
A260 ssDNA = 37 µg/ml
A260 ssRNA = 40 µg/ml
Conversion factors
When using a 10 mm path lengthPath length
In chemistry, the path length is defined as the distance that light travels through a sample in an analytical cell. Typically, a sample cell is made of quartz, glass, or a plastic rhombic cuvette with a volume typically ranging from 0.1 mL to 10 mL or larger used in a spectrophotometer. For the...
, simply multiply the OD by the conversion factor
Conversion factor
A conversion factor changes something to a different version or form. A factor is something that brings results or a cause, while conversion is an action of changing the "version" of a thing....
to determine the concentration.
Example, a 2.0 OD dsDNA sample corresponds to a sample with a 100 ug/ml concentration.
When using a path length that is shorter than 10mm, the resultant OD will be reduced by a factor of 10/path length. Using the example above with a 3 mm path length, the OD for the 100 ug/ml sample would be reduced to 0.6. To normalize the concentration to a 10mm equivalent, the following is done:
0.6 OD X (10/3) * 50 ug/ml=100 ug/ml
Most spectrophotometers, like the AQ-07 Nucleic Acid Meter, allow selection of the nucleic acid type and path length such that resultant concentration is normalized to the 10mm path length which is based on the principles of Beer's law.
A260 as quantity measure
The "A260 unit" is used a quantity measure for nucleic acids. One A260 unit is the amount of nucleic acid contained in 1 mL and producing a OD of 1. The same conversion factors apply, and therefore, in such contexts:1 A260 unit dsDNA = 50 µg
1 A260 unit ssDNA = 37 µg
1 A260 unit ssRNA = 40 µg
Determination of sample purity
The secondary benefit of using spectrophotometric analysis for nucleic acid quantitation is the ability to determine sample purity using the 260 nm:280 nm calculation. A pure DNA sample will yield an A260/280 of approximately 1.8. For RNA, a pure sample will yield anA260/280 of approximately 2.0. These ratios are commonly used to assess the amount of protein contamination that is left from the nucleic acid isolation process since proteins absorb at 280 nm.Fluorescence tagging
In some cases, scientists elect to quantitate DNA and RNA using fluorescence taggingFluorescent tag
In molecular biology and biotechnology, a fluorescent tag is a part of a molecule that researchers have attached chemically to aid in detection of the molecule to which it has been attached. The tag is some kind of fluorescent molecule...
methods with dye
Dye
A dye is a colored substance that has an affinity to the substrate to which it is being applied. The dye is generally applied in an aqueous solution, and requires a mordant to improve the fastness of the dye on the fiber....
s that are highly specific to DNA or RNA. The benefit of fluorescence quantitation of DNA and RNA is the improved sensitivity over spectrophotometric analysis
Spectrophotometry
In chemistry, spectrophotometry is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength...
. Although, that increase in sensitivity comes at the cost of a higher price per sample and a lengthier sample preparation process. Additionally, there is not currently a fluorescence method to determine protein contamination of a DNA sample that is similar to the 260 nm/280 nm spectrophotometric version.