Agarose gel electrophoresis

An agarose gel cast in tray, to be used for gel electrophoresis

Agarose gel is a three-dimensional matrix formed of helical agarose molecules in supercoiled bundles that are aggregated into three-dimensional structures with channels and pores through which biomolecules can pass.^[3] The 3-D structure is held together with hydrogen bonds and can therefore be disrupted by heating back to a liquid state. The melting temperature is different from the gelling temperature, depending on the sources, agarose gel has a gelling temperature of 35–42 °C and a melting temperature of 85–95 °C. Low-melting and low-gelling agaroses made through chemical modifications are also available.

Agarose gel has large pore size and good gel strength, making it suitable as an anticonvection medium for the electrophoresis of DNA and large protein molecules. The pore size of a 1% gel has been estimated from 100 nm to 200–500 nm,^[4][5] and its gel strength allows gels as dilute as 0.15% to form a slab for gel electrophoresis.^[6] Low-concentration gels (0.1–0.2%) however are fragile and therefore hard to handle. Agarose gel has lower resolving power than polyacrylamide gel for DNA but has a greater range of separation, and is therefore used for DNA fragments of usually 50–20,000 bp in size. The limit of resolution for standard agarose gel electrophoresis is around 750 kb, but resolution of over 6 Mb is possible with pulsed field gel electrophoresis (PFGE).^[7] It can also be used to separate large proteins, and it is the preferred matrix for the gel electrophoresis of particles with effective radii larger than 5–10 nm. A 0.9% agarose gel has pores large enough for the entry of bacteriophage T4.^[6]

The agarose polymer contains charged groups, in particular pyruvate and sulphate.^[8] These negatively charged groups create a flow of water in the opposite direction to the movement of DNA in a process called electroendosmosis (EEO), and can therefore retard the movement of DNA and cause blurring of bands. Higher concentration gels would have higher electroendosmotic flow. Low EEO agarose is therefore generally preferred for use in agarose gel electrophoresis of nucleic acids, but high EEO agarose may be used for other purposes. The lower sulphate content of low EEO agarose, particularly low-melting point (LMP) agarose, is also beneficial in cases where the DNA extracted from gel is to be used for further manipulation as the presence of contaminating sulphates may affect some subsequent procedures, such as ligation and PCR. Zero EEO agaroses however are undesirable for some applications as they may be made by adding positively charged groups and such groups can affect subsequent enzyme reactions.^[9] Electroendosmosis is a reason agarose is used in preference to agar as the agaropectin component in agar contains a significant amount of negatively charged sulphate and carboxyl groups. The removal of agaropectin in agarose substantially reduces the EEO, as well as reducing the non-specific adsorption of biomolecules to the gel matrix. However, for some applications such as the electrophoresis of serum proteins, a high EEO may be desirable, and agaropectin may be added in the gel used.^[10]

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Agarose

Agarose

Agarose is a heteropolysaccharide, generally extracted from certain red seaweed. It is a linear polymer made up of the repeating unit of agarobiose, which is a disaccharide made up of D-galactose and 3,6-anhydro-L-galactopyranose. Agarose is one of the two principal components of agar, and is purified from agar by removing agar's other component, agaropectin.

Gel electrophoresis of nucleic acids

Gel electrophoresis of nucleic acids

Nucleic acid electrophoresis is an analytical technique used to separate DNA or RNA fragments by size and reactivity. Nucleic acid molecules which are to be analyzed are set upon a viscous medium, the gel, where an electric field induces the nucleic acids to migrate toward the anode. The separation of these fragments is accomplished by exploiting the mobilities with which different sized molecules are able to pass through the gel. Longer molecules migrate more slowly because they experience more resistance within the gel. Because the size of the molecule affects its mobility, smaller fragments end up nearer to the anode than longer ones in a given period. After some time, the voltage is removed and the fragmentation gradient is analyzed. For larger separations between similar sized fragments, either the voltage or run time can be increased. Extended runs across a low voltage gel yield the most accurate resolution. Voltage is, however, not the sole factor in determining electrophoresis of nucleic acids.

Ligation (molecular biology)

Ligation (molecular biology)

Ligation is the joining of two nucleic acid fragments through the action of an enzyme. It is an essential laboratory procedure in the molecular cloning of DNA, whereby DNA fragments are joined to create recombinant DNA molecules (such as when a foreign DNA fragment is inserted into a plasmid). The ends of DNA fragments are joined by the formation of phosphodiester bonds between the 3'-hydroxyl of one DNA terminus with the 5'-phosphoryl of another. RNA may also be ligated similarly. A co-factor is generally involved in the reaction, and this is usually ATP or NAD+. Eukaryotic cells ligases belong to ATP type, and NAD+ - dependent are found in bacteria (e.g. E. coli).

Polymerase chain reaction

Polymerase chain reaction

The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it to a large enough amount to study in detail. PCR was invented in 1983 by the American biochemist Kary Mullis at Cetus Corporation; Mullis and biochemist Michael Smith, who had developed other essential ways of manipulating DNA, were jointly awarded the Nobel Prize in Chemistry in 1993.

Agar

Agar

Agar, or agar-agar, is a jelly-like substance consisting of polysaccharides obtained from the cell walls of some species of red algae, primarily from "ogonori" (Gracilaria) and "tengusa" (Gelidiaceae). As found in nature, agar is a mixture of two components, the linear polysaccharide agarose and a heterogeneous mixture of smaller molecules called agaropectin. It forms the supporting structure in the cell walls of certain species of algae and is released on boiling. These algae are known as agarophytes, belonging to the Rhodophyta phylum. The processing of food-grade agar removes the agaropectin, and the commercial product is essentially pure agarose.

Agaropectin

Agaropectin

Agaropectin is one of the two main components of agar and mainly consists of D-glucuronic acid and pyruvic acid.

Factors affecting migration of nucleic acid in gel

Gels of plasmid preparations usually show a major band of supercoiled DNA with other fainter bands in the same lane. Note that by convention DNA gel is displayed with smaller DNA fragments nearer to the bottom of the gel. This is because historically DNA gels were run vertically and the smaller DNA fragments move downwards faster.

A number of factors can affect the migration of nucleic acids: the dimension of the gel pores (gel concentration), size of DNA being electrophoresed, the voltage used, the ionic strength of the buffer, and the concentration of intercalating dye such as ethidium bromide if used during electrophoresis.^[11]

Smaller molecules travel faster than larger molecules in gel, and double-stranded DNA moves at a rate that is inversely proportional to the logarithm of the number of base pairs. This relationship however breaks down with very large DNA fragments, and separation of very large DNA fragments requires the use of pulsed field gel electrophoresis (PFGE), which applies alternating current from different directions and the large DNA fragments are separated as they reorient themselves with the changing field.^[12]

For standard agarose gel electrophoresis, larger molecules are resolved better using a low concentration gel while smaller molecules separate better at high concentration gel. Higher concentration gels, however, require longer run times (sometimes days).

The movement of the DNA may be affected by the conformation of the DNA molecule, for example, supercoiled DNA usually moves faster than relaxed DNA because it is tightly coiled and hence more compact. In a normal plasmid DNA preparation, multiple forms of DNA may be present.^[13] Gel electrophoresis of the plasmids would normally show the negatively supercoiled form as the main band, while nicked DNA (open circular form) and the relaxed closed circular form appears as minor bands. The rate at which the various forms move however can change using different electrophoresis conditions,^[14] and the mobility of larger circular DNA may be more strongly affected than linear DNA by the pore size of the gel.^[15]

Ethidium bromide which intercalates into circular DNA can change the charge, length, as well as the superhelicity of the DNA molecule, therefore its presence in gel during electrophoresis can affect its movement. For example, the positive charge of ethidium bromide can reduce the DNA movement by 15%.^[12] Agarose gel electrophoresis can be used to resolve circular DNA with different supercoiling topology.^[16]

DNA damage due to increased cross-linking will also reduce electrophoretic DNA migration in a dose-dependent way.^[17][18]

The rate of migration of the DNA is proportional to the voltage applied, i.e. the higher the voltage, the faster the DNA moves. The resolution of large DNA fragments however is lower at high voltage. The mobility of DNA may also change in an unsteady field – in a field that is periodically reversed, the mobility of DNA of a particular size may drop significantly at a particular cycling frequency.^[4] This phenomenon can result in band inversion in field inversion gel electrophoresis (FIGE), whereby larger DNA fragments move faster than smaller ones.

Migration anomalies

• "Smiley" gels - this edge effect is caused when the voltage applied is too high for the gel concentration used.^[19]
• Overloading of DNA - overloading of DNA slows down the migration of DNA fragments.
• Contamination - presence of impurities, such as salts or proteins can affect the movement of the DNA.

Mechanism of migration and separation

The negative charge of its phosphate backbone moves the DNA towards the positively charged anode during electrophoresis. However, the migration of DNA molecules in solution, in the absence of a gel matrix, is independent of molecular weight during electrophoresis.^[4][20] The gel matrix is therefore responsible for the separation of DNA by size during electrophoresis, and a number of models exist to explain the mechanism of separation of biomolecules in gel matrix. A widely accepted one is the Ogston model which treats the polymer matrix as a sieve. A globular protein or a random coil DNA moves through the interconnected pores, and the movement of larger molecules is more likely to be impeded and slowed down by collisions with the gel matrix, and the molecules of different sizes can therefore be separated in this sieving process.^[4]

The Ogston model however breaks down for large molecules whereby the pores are significantly smaller than size of the molecule. For DNA molecules of size greater than 1 kb, a reptation model (or its variants) is most commonly used. This model assumes that the DNA can crawl in a "snake-like" fashion (hence "reptation") through the pores as an elongated molecule. A biased reptation model applies at higher electric field strength, whereby the leading end of the molecule become strongly biased in the forward direction and pulls the rest of the molecule along.^[21] Real-time fluorescence microscopy of stained molecules, however, showed more subtle dynamics during electrophoresis, with the DNA showing considerable elasticity as it alternately stretching in the direction of the applied field and then contracting into a ball, or becoming hooked into a U-shape when it gets caught on the polymer fibres.^[22][23]

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Gel electrophoresis of nucleic acids

Gel electrophoresis of nucleic acids

Nucleic acid electrophoresis is an analytical technique used to separate DNA or RNA fragments by size and reactivity. Nucleic acid molecules which are to be analyzed are set upon a viscous medium, the gel, where an electric field induces the nucleic acids to migrate toward the anode. The separation of these fragments is accomplished by exploiting the mobilities with which different sized molecules are able to pass through the gel. Longer molecules migrate more slowly because they experience more resistance within the gel. Because the size of the molecule affects its mobility, smaller fragments end up nearer to the anode than longer ones in a given period. After some time, the voltage is removed and the fragmentation gradient is analyzed. For larger separations between similar sized fragments, either the voltage or run time can be increased. Extended runs across a low voltage gel yield the most accurate resolution. Voltage is, however, not the sole factor in determining electrophoresis of nucleic acids.

Chemical structure

Chemical structure

A chemical structure determination includes a chemist's specifying the molecular geometry and, when feasible and necessary, the electronic structure of the target molecule or other solid. Molecular geometry refers to the spatial arrangement of atoms in a molecule and the chemical bonds that hold the atoms together, and can be represented using structural formulae and by molecular models; complete electronic structure descriptions include specifying the occupation of a molecule's molecular orbitals. Structure determination can be applied to a range of targets from very simple molecules, to very complex ones.

DNA supercoil

DNA supercoil

DNA supercoiling refers to the amount of twist in a particular DNA strand, which determines the amount of strain on it. A given strand may be "positively supercoiled" or "negatively supercoiled". The amount of a strand’s supercoiling affects a number of biological processes, such as compacting DNA and regulating access to the genetic code. Certain enzymes, such as topoisomerases, change the amount of DNA supercoiling to facilitate functions such as DNA replication and transcription. The amount of supercoiling in a given strand is described by a mathematical formula that compares it to a reference state known as "relaxed B-form" DNA.

Cross-link

Cross-link

In chemistry and biology a cross-link is a bond or a short sequence of bonds that links one polymer chain to another. These links may take the form of covalent bonds or ionic bonds and the polymers can be either synthetic polymers or natural polymers.

Random coil

Random coil

A random coil is a polymer conformation where the monomer subunits are oriented randomly while still being bonded to adjacent units. It is not one specific shape, but a statistical distribution of shapes for all the chains in a population of macromolecules. The conformation's name is derived from the idea that, in the absence of specific, stabilizing interactions, a polymer backbone will "sample" all possible conformations randomly. Many linear, unbranched homopolymers — in solution, or above their melting temperatures — assume (approximate) random coils.

Reptation

Reptation

A peculiarity of thermal motion of very long linear macromolecules in entangled polymer melts or concentrated polymer solutions is reptation. Derived from the word reptile, reptation suggests the movement of entangled polymer chains as being analogous to snakes slithering through one another. Pierre-Gilles de Gennes introduced the concept of reptation into polymer physics in 1971 to explain the dependence of the mobility of a macromolecule on its length. Reptation is used as a mechanism to explain viscous flow in an amorphous polymer. Sir Sam Edwards and Masao Doi later refined reptation theory. Similar phenomena also occur in proteins.

The details of an agarose gel electrophoresis experiment may vary depending on methods, but most follow a general procedure.

Casting of gel

Loading DNA samples into the wells of an agarose gel using a multi-channel pipette.

The gel is prepared by dissolving the agarose powder in an appropriate buffer, such as TAE or TBE, to be used in electrophoresis.^[12] The agarose is dispersed in the buffer before heating it to near-boiling point, but avoid boiling. The melted agarose is allowed to cool sufficiently before pouring the solution into a cast as the cast may warp or crack if the agarose solution is too hot. A comb is placed in the cast to create wells for loading sample, and the gel should be completely set before use.

The concentration of gel affects the resolution of DNA separation. The agarose gel is composed of microscopic pores through which the molecules travel, and there is an inverse relationship between the pore size of the agarose gel and the concentration – pore size decreases as the density of agarose fibers increases. High gel concentration improves separation of smaller DNA molecules, while lowering gel concentration permits large DNA molecules to be separated. The process allows fragments ranging from 50 base pairs to several mega bases to be separated depending on the gel concentration used.^[24] The concentration is measured in weight of agarose over volume of buffer used (g/ml). For a standard agarose gel electrophoresis, a 0.8% gel gives good separation or resolution of large 5–10kb DNA fragments, while 2% gel gives good resolution for small 0.2–1kb fragments. 1% gels is often used for a standard electrophoresis.^[25] High percentage gels are often brittle and may not set evenly, while low percentage gels (0.1-0.2%) are fragile and not easy to handle. Low-melting-point (LMP) agarose gels are also more fragile than normal agarose gel. Low-melting point agarose may be used on its own or simultaneously with standard agarose for the separation and isolation of DNA.^[26] PFGE and FIGE are often done with high percentage agarose gels.

Loading of samples

Once the gel has set, the comb is removed, leaving wells where DNA samples can be loaded. Loading buffer is mixed with the DNA sample before the mixture is loaded into the wells. The loading buffer contains a dense compound, which may be glycerol, sucrose, or Ficoll, that raises the density of the sample so that the DNA sample may sink to the bottom of the well.^[12] If the DNA sample contains residual ethanol after its preparation, it may float out of the well. The loading buffer also includes colored dyes such as xylene cyanol and bromophenol blue used to monitor the progress of the electrophoresis. The DNA samples are loaded using a pipette.

Electrophoresis

Agarose gel slab in electrophoresis tank with bands of dyes indicating progress of the electrophoresis. The DNA moves towards anode.

Agarose gel electrophoresis is most commonly done horizontally in a subaquaeous mode whereby the slab gel is completely submerged in buffer during electrophoresis. It is also possible, but less common, to perform the electrophoresis vertically, as well as horizontally with the gel raised on agarose legs using an appropriate apparatus.^[27] The buffer used in the gel is the same as the running buffer in the electrophoresis tank, which is why electrophoresis in the subaquaeous mode is possible with agarose gel.

For optimal resolution of DNA greater than 2 kb in size in standard gel electrophoresis, 5 to 8 V/cm is recommended (the distance in cm refers to the distance between electrodes, therefore this recommended voltage would be 5 to 8 multiplied by the distance between the electrodes in cm).^[14] Voltage may also be limited by the fact that it heats the gel and may cause the gel to melt if it is run at high voltage for a prolonged period, especially if the gel used is LMP agarose gel. Too high a voltage may also reduce resolution, as well as causing band streaking for large DNA molecules. Too low a voltage may lead to broadening of band for small DNA fragments due to dispersion and diffusion.^[28]

Since DNA is not visible in natural light, the progress of the electrophoresis is monitored using colored dyes. Xylene cyanol (light blue color) comigrates large DNA fragments, while Bromophenol blue (dark blue) comigrates with the smaller fragments. Less commonly used dyes include Cresol Red and Orange G which migrate ahead of bromophenol blue. A DNA marker is also run together for the estimation of the molecular weight of the DNA fragments. Note however that the size of a circular DNA like plasmids cannot be accurately gauged using standard markers unless it has been linearized by restriction digest, alternatively a supercoiled DNA marker may be used.

Staining and visualization

The gel with UV illumination: DNA stained with ethidium bromide appears as glowing orange bands.

DNA as well as RNA are normally visualized by staining with ethidium bromide, which intercalates into the major grooves of the DNA and fluoresces under UV light. The intercalation depends on the concentration of DNA and thus, a band with high intensity will indicate a higher amount of DNA compared to a band of less intensity.^[12] The ethidium bromide may be added to the agarose solution before it gels, or the DNA gel may be stained later after electrophoresis. Destaining of the gel is not necessary but may produce better images. Other methods of staining are available; examples are SYBR Green, GelRed, methylene blue, brilliant cresyl blue, Nile blue sulphate, and crystal violet.^[29] SYBR Green, GelRed and other similar commercial products are sold as safer alternatives to ethidium bromide as it has been shown to be mutagenic in Ames test, although the carcinogenicity of ethidium bromide has not actually been established. SYBR Green requires the use of a blue-light transilluminator. DNA stained with crystal violet can be viewed under natural light without the use of a UV transilluminator which is an advantage, however it may not produce a strong band.

When stained with ethidium bromide, the gel is viewed with an ultraviolet (UV) transilluminator. The UV light excites the electrons within the aromatic ring of ethidium bromide, and once they return to the ground state, light is released, making the DNA and ethidium bromide complex fluoresce.^[12] Standard transilluminators use wavelengths of 302/312-nm (UV-B), however exposure of DNA to UV radiation for as little as 45 seconds can produce damage to DNA and affect subsequent procedures, for example reducing the efficiency of transformation, in vitro transcription, and PCR.^[30] Exposure of DNA to UV radiation therefore should be limited. Using a higher wavelength of 365 nm (UV-A range) causes less damage to the DNA but also produces much weaker fluorescence with ethidium bromide. Where multiple wavelengths can be selected in the transilluminator, shorter wavelength can be used to capture images, while longer wavelength should be used if it is necessary to work on the gel for any extended period of time.

The transilluminator apparatus may also contain image capture devices, such as a digital or polaroid camera, that allow an image of the gel to be taken or printed.

For gel electrophoresis of protein, the bands may be visualised with Coomassie or silver stains.

Cutting out agarose gel slices. Protective equipment must be worn when using UV transilluminator.

Downstream procedures

The separated DNA bands are often used for further procedures, and a DNA band may be cut out of the gel as a slice, dissolved and purified. Contaminants however may affect some downstream procedures such as PCR, and low melting point agarose may be preferred in some cases as it contains fewer of the sulphates that can affect some enzymatic reactions. The gels may also be used for blotting techniques.

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Ficoll

Ficoll

Ficoll is a neutral, highly branched, high-mass, hydrophilic polysaccharide which dissolves readily in aqueous solutions. Ficoll radii range from 2 to 7 nm. It is prepared by reaction of the polysaccharide with epichlorohydrin. Ficoll is a registered trademark owned by GE Healthcare companies.

Bromophenol blue

Bromophenol blue

Bromophenol blue, albutest is used as a pH indicator, an electrophoretic color marker, and a dye. It can be prepared by slowly adding excess bromine to a hot solution of phenolsulfonphthalein in glacial acetic acid.

Pipette

Pipette

A pipette is a laboratory tool commonly used in chemistry, biology and medicine to transport a measured volume of liquid, often as a media dispenser. Pipettes come in several designs for various purposes with differing levels of accuracy and precision, from single piece glass pipettes to more complex adjustable or electronic pipettes. Many pipette types work by creating a partial vacuum above the liquid-holding chamber and selectively releasing this vacuum to draw up and dispense liquid. Measurement accuracy varies greatly depending on the instrument.

Cresol Red

Cresol Red

Cresol red is a triarylmethane dye frequently used for monitoring the pH in aquaria.

Orange G

Orange G

Orange G also called C.I. 16230, Acid Orange 10, or orange gelb is a synthetic azo dye used in histology in many staining formulations. It usually comes as a disodium salt. It has the appearance of orange crystals or powder.

Ethidium bromide

Ethidium bromide

Ethidium bromide is an intercalating agent commonly used as a fluorescent tag in molecular biology laboratories for techniques such as agarose gel electrophoresis. It is commonly abbreviated as EtBr, which is also an abbreviation for bromoethane. To avoid confusion, some laboratories have used the abbreviation EthBr for this salt. When exposed to ultraviolet light, it will fluoresce with an orange colour, intensifying almost 20-fold after binding to DNA. Under the name homidium, it has been commonly used since the 1950s in veterinary medicine to treat trypanosomiasis in cattle. The high incidence of antimicrobial resistance makes this treatment impractical in some areas, where the related isometamidium chloride is used instead. Despite its reputation as a mutagen, it is relatively safe to handle.

GelRed

GelRed

GelRed is an intercalating nucleic acid stain used in molecular genetics for agarose gel DNA electrophoresis. GelRed structurally consists of two ethidium subunits that are bridged by a linear oxygenated spacer.

Methylene blue

Methylene blue

Methylthioninium chloride, commonly called methylene blue, is a salt used as a dye and as a medication. Methylene blue is a thiazine dye. As a medication, it is mainly used to treat methemoglobinemia by converting/chemically reducing the ferric iron in hemoglobin to ferrous iron. Specifically, it is used to treat methemoglobin levels that are greater than 30% or in which there are symptoms despite oxygen therapy. It has previously been used for treating cyanide poisoning and urinary tract infections, but this use is no longer recommended.

Brilliant cresyl blue

Brilliant cresyl blue

Brilliant cresyl blue is a supravital stain used for counting reticulocytes. It is classified as an oxazine dye. N95 dust masks, eye shields, and gloves must all be worn when handling the chemical.

Nile blue

Nile blue

Nile blue is a stain used in biology and histology. It may be used with live or fixed cells, and imparts a blue colour to cell nuclei.

Crystal violet

Crystal violet

Crystal violet or gentian violet, also known as methyl violet 10B or hexamethyl pararosaniline chloride, is a triarylmethane dye used as a histological stain and in Gram's method of classifying bacteria. Crystal violet has antibacterial, antifungal, and anthelmintic (vermicide) properties and was formerly important as a topical antiseptic. The medical use of the dye has been largely superseded by more modern drugs, although it is still listed by the World Health Organization.

Ames test

Ames test

The Ames test is a widely employed method that uses bacteria to test whether a given chemical can cause mutations in the DNA of the test organism. More formally, it is a biological assay to assess the mutagenic potential of chemical compounds. A positive test indicates that the chemical is mutagenic and therefore may act as a carcinogen, because cancer is often linked to mutation. The test serves as a quick and convenient assay to estimate the carcinogenic potential of a compound because standard carcinogen assays on mice and rats are time-consuming and expensive. However, false-positives and false-negatives are known.

In general, the ideal buffer should have good conductivity, produce less heat and have a long life.^[31] There are a number of buffers used for agarose electrophoresis; common ones for nucleic acids include Tris/Acetate/EDTA (TAE) and Tris/Borate/EDTA (TBE). The buffers used contain EDTA to inactivate many nucleases which require divalent cation for their function. The borate in TBE buffer can be problematic as borate can polymerize, and/or interact with cis diols such as those found in RNA. TAE has the lowest buffering capacity, but it provides the best resolution for larger DNA. This means a lower voltage and more time, but a better product.

Many other buffers have been proposed, e.g. lithium borate (LB), iso electric histidine, pK matched goods buffers, etc.; in most cases the purported rationale is lower current (less heat) and or matched ion mobilities, which leads to longer buffer life. Tris-phosphate buffer has high buffering capacity but cannot be used if DNA extracted is to be used in phosphate sensitive reaction. LB is relatively new and is ineffective in resolving fragments larger than 5 kbp; However, with its low conductivity, a much higher voltage could be used (up to 35 V/cm), which means a shorter analysis time for routine electrophoresis. As low as one base pair size difference could be resolved in 3% agarose gel with an extremely low conductivity medium (1 mM lithium borate).^[32]

Other buffering system may be used in specific applications, for example, barbituric acid-sodium barbiturate or Tris-barbiturate buffers may be used for in agarose gel electrophoresis of proteins, for example in the detection of abnormal distribution of proteins.^[33]

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TAE buffer

TAE buffer

TAE buffer is a buffer solution containing a mixture of Tris base, acetic acid and EDTA.

TBE buffer

TBE buffer

TBE or Tris/Borate/EDTA, is a buffer solution containing a mixture of Tris base, boric acid and EDTA.

LB buffer

LB buffer

LB buffer, also known as lithium borate buffer, is a buffer solution used in agarose electrophoresis, typically for the separation of nucleic acids such as DNA and RNA. It is made up of Lithium borate.

Barbital

Barbital

Barbital, marketed under the brand names Veronal for the pure acid and Medinal for the sodium salt, was the first commercially available barbiturate. It was used as a sleeping aid (hypnotic) from 1903 until the mid-1950s. The chemical names for barbital are diethylmalonyl urea or diethylbarbituric acid; hence, the sodium salt is known also as sodium diethylbarbiturate.

• Estimation of the size of DNA molecules following digestion with restriction enzymes, e.g., in restriction mapping of cloned DNA.
• Estimation of the DNA concentration by comparing the intensity of the nucleic acid band with the corresponding band of the size marker.^[34]
• Analysis of products of a polymerase chain reaction (PCR), e.g., in molecular genetic diagnosis or genetic fingerprinting
• Separation of DNA fragments for extraction and purification.
• Separation of restricted genomic DNA prior to Southern transfer, or of RNA prior to Northern transfer.
• Separation of proteins, for example, screening of protein abnormalities in clinical chemistry.^[35]

Agarose gels are easily cast and handled compared to other matrices and nucleic acids are not chemically altered during electrophoresis. Samples are also easily recovered. After the experiment is finished, the resulting gel can be stored in a plastic bag in a refrigerator.

Electrophoresis is performed in buffer solutions to reduce pH changes due to the electric field, which is important because the charge of DNA and RNA depends on pH, but running for too long can exhaust the buffering capacity of the solution. Further, different preparations of genetic material may not migrate consistently with each other, for morphological or other reasons.

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Restriction enzyme

Restriction enzyme

A restriction enzyme, restriction endonuclease, REase, ENase or restrictase is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class of the broader endonuclease group of enzymes. Restriction enzymes are commonly classified into five types, which differ in their structure and whether they cut their DNA substrate at their recognition site, or if the recognition and cleavage sites are separate from one another. To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone of the DNA double helix.

Restriction map

Restriction map

A restriction map is a map of known restriction sites within a sequence of DNA. Restriction mapping requires the use of restriction enzymes. In molecular biology, restriction maps are used as a reference to engineer plasmids or other relatively short pieces of DNA, and sometimes for longer genomic DNA. There are other ways of mapping features on DNA for longer length DNA molecules, such as mapping by transduction.

Polymerase chain reaction

Polymerase chain reaction

The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it to a large enough amount to study in detail. PCR was invented in 1983 by the American biochemist Kary Mullis at Cetus Corporation; Mullis and biochemist Michael Smith, who had developed other essential ways of manipulating DNA, were jointly awarded the Nobel Prize in Chemistry in 1993.

Preimplantation genetic diagnosis

Preimplantation genetic diagnosis

Preimplantation genetic diagnosis is the genetic profiling of embryos prior to implantation, and sometimes even of oocytes prior to fertilization. PGD is considered in a similar fashion to prenatal diagnosis. When used to screen for a specific genetic disease, its main advantage is that it avoids selective abortion, as the method makes it highly likely that the baby will be free of the disease under consideration. PGD thus is an adjunct to assisted reproductive technology, and requires in vitro fertilization (IVF) to obtain oocytes or embryos for evaluation. Embryos are generally obtained through blastomere or blastocyst biopsy. The latter technique has proved to be less deleterious for the embryo, therefore it is advisable to perform the biopsy around day 5 or 6 of development.

Southern blot

Southern blot

Southern blot is a method used for detection and quantification of a specific DNA sequence in DNA samples. This method is used in molecular biology. Briefly, purified DNA from a biological sample is digested with restriction enzymes, and the resulting DNA fragments are separated by using an electric current to move them through a sieve-like gel or matrix, which allows smaller fragments to move faster than larger fragments. The DNA fragments are transferred out of the gel or matrix onto a solid membrane, which is then exposed to a DNA probe labeled with a radioactive, fluorescent, or chemical tag. The tag allows any DNA fragments containing complementary sequences with the DNA probe sequence to be visualized within the Southern blot.

Clinical chemistry

Clinical chemistry

Clinical chemistry is the area of chemistry that is generally concerned with analysis of bodily fluids for diagnostic and therapeutic purposes. It is an applied form of biochemistry.