Pharos university Faculty of Allied Medical SCIENCE Clinical Laboratory Instrumentation (MELI-201) Dr. Tarek El Sewedy

Lecture 4 Electrophoresis And Spectrophotometers

Intended Learning Outcomes Students will learn : 1.Electrophoresis principles. 2.Electrophoresis techniques. 3.Spectrophotometry

Lecture content  Electrophoresis principles.  DNA Electrophoresis.  Protein Electrophoresis  Spectrophotometers

Electrophoresis

 A method of separating molecules from a mixture of similar molecules using electrical current.  The electric current is passed through a porous solid material called gel containing the mixture, and each molecule travels through the gel at a different rate, depending on its size.  Gels are either Agarose (for DNA) or polyacrylamide gels (for Proteins).  Electrophoresis is most commonly used to identify DNA fragments and proteins  It can be followed by a technique called blotting to specifically identify a particular DNA segment (Southern blotting) or certain type of protein (Western blotting) or RNA (Northern Blotting)

1. DNA electrophoresis

How gel DNA electrophoresis works 1  First a gel is prepared, DNA Gels are made of Agarose, a seaweed extract similar to gelatin. The finished gel has a consistency similar to very firm jello. This consistency offers resistance to the pieces of DNA as they try to move through the gel. The gel is prepared with wells at one end so that DNA samples can be loaded into the gel.

 Once the DNA samples are loaded onto the gel, an electric current is applied to the gel. DNA is negatively charged due to all the phosphate groups in the backbone of DNA. Thus, DNA will move towards the positive electrode. How gel electrophoresis works 2

 As the pieces of DNA move through the gel, they will meet with resistance. Larger pieces of DNA will have more difficulty moving through the gel than smaller fragments. Thus, larger fragments will move slower than smaller fragments. This allows separation of different DNA fragments depending on size difference. How gel electrophoresis works 3

Electrophoretic mobility of DNA

Summary of DNA electrophoresis

2. Protein Electrophoresis

 Movement of proteins in polyacrylamide gels is more complex. Standard polyacrylamide gels (PAGE) separate the proteins down the gel based on differences in the protein’s degree of electrical charge, shape, and size.  A type of treatment called denaturation is used in SDS polyacrylamide gels (SDS-PAGE). The proteins are heated in a solution called sodium dodecyl sulfate or SDS. This causes the proteins to have similar charges and shapes. Therefore, separation is based on size. Again, smaller molecules move more quickly to the Anode on the lower portion of the gel.

Protein Electrophoresis

Protein Electrophoresis Gels

Electrophoresis in clinical Labs  The electrophoretic graph is the interpretation of protein electrophoresis and includes a variety of proteins

Uses of DNA Gel Electrophoresis  Gel electrophoresis is used to provide genetic information in a wide range of data fields. Human DNA can be analyzed to provide evidence in criminal cases, to diagnose genetic diseases, and to solve paternity cases. Samples can be obtained from any DNA-containing tissue or body fluid, including tissue cells, blood, skin, hair, and semen. In many analyses, polymerase chain reaction (PCR) is used to amplify specific regions of DNA that are known to vary among individuals. A person’s “DNA fingerprint” or “DNA profile” is constructed by using gel electrophoresis to separate the DNA fragments from several of these highly variable regions.

Spectrophotometers  Spectroscopy uses light to identify and determine the concentration of a particular chemical in a solution.  The term spectroscopy refers to the observation (scopy) of various wavelengths of light (spectro).  It makes use of the ultraviolet, visible, and infrared regions of light spectrum.  Humans perceive different types of light as color. However, scientists measure light as wavelength.

 Spectrophotometers are found in every clinical laboratory. They are important for identifying biological molecules. These instruments are also useful for determining the concentration and purity of almost all biological molecules.  The basic spectrophotometer is composed of a light source, monochromator, sample holder, photodetector, and readout.

 Most spectroscopy uses a light source made up of a lamp that produces visible light very much like a household light bulb. These lamps provide white light which is made up of the whole visible light spectrum.  Infrared or ultraviolet light lamps can also be used as a light source. The type of light used depends on the nature of the chemical testing being performed.  Lamps usually have to be warmed up and maintained at a certain temperature to ensure consistent lighting during each use.  An improperly operating lamp can give the wrong information without the operator being aware of any errors

 spectrophotometers provide a pure beam of light consisting of a few wavelengths that is then transmitted to the chemical sample.  This pure beam of light is produced by using a device called a monochromator.  A monochromator is an adjustable crystal, filter, or mirror that isolates portions of the light spectrum by separating the light into its component wavelengths.  The range of wavelengths provided by a monochromator is called the bandwidth.

 The sample holder is an opening in the spectrophotometer that places the sample between the beam path and the photodetector.  The sample holder grips a special container called a cuvette. Cuvettes are made of glass, plastic, quartz. The type of cuvette being used is determined by the wavelength of the beam. Ultraviolet light spectroscopy requires quartz cuvettes because glass and plastic interfere with the passage of ultraviolet light to the sample.

 The photodetector, measures light: 1.Absorbance: amount of light absorbed by the sample. 2.Transmittance: amount the light passed through the sample. 3.Fluorescence: amount of glow given off by a sample that is exposed to a particular wavelength of light.  The photodetector converts the light into an electrical signal that is displayed as a measurement on the readout.  The readout usually displays the measurement as a number.

Assignment  Abdulrahman Samir: is selected to make the assignment Different applications of Electrophoresis  The Assignment should be delivered before next lecture

Study questions  Mentions 3 different applications of Electrophoresis  Mention the main difference between DNA and Protein electrophoresis

Suggesting reading  Encyclopedia of Medical Devices and Instrumentation, 2nd ed. New York: Wiley, 2006