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DNA ladder

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DNA ladder

Not to be confused with molecular marker, which is a genetic marker.

A molecular-weight size marker, also referred to as a protein ladder or DNA ladder, is a set of standards that are used to identify the approximate size of a molecule run on a gel during electrophoresis, using the principle that molecular weight is inversely proportional to migration rate through a gel matrix. Therefore when used in gel electrophoresis, markers effectively provide a logarithmic scale by which to estimate the size of the other fragments (providing the fragment sizes of the marker are known).

Protein and DNA markers with pre-determined fragment sizes and concentrations are commercially available. These can be run in either agarose or polyacrylamide gels. The markers are loaded in lanes adjacent to sample lanes before the commencement of the run.

Development

Although the concept of molecular-weight markers has been retained, techniques of development have varied throughout the years. New inventions of molecular-weight markers are distributed in kits specific to the marker's type.

DNA markers

An early problem in the development of markers was achieving high resolution throughout the entire length of the ladder.[1] Depending on the running conditions of gel electrophoresis, fragments may have been compressed, disrupting clarity. To address this issue, a kit for Southern Blot analysis was developed 1990, providing the first marker to combine target DNA and probe DNA. This technique took advantage of logarithmic spacing, and could be used to identify target bands ranging over a length of 20,000 nucleotides.[2]

Protein markers

Previously, protein markers had been developed using a variety of whole proteins. The development of a kit including a molecular-weight size ladder based on protein fragments began in 1993. This protein ladder, composed of 49 different amino acid sequences, included multidomain proteins, and allowed for the analysis of proteins cleaved at different sites.[3]

Current technique improvements in protein ladders involve the use of auto-development. The first auto-developed regularly-weight protein ladder was invented in 2012.[4]

DNA markers

A commonly used DNA molecular weight marker is the genome of the Lambda phage following digestion using the restriction enzyme HindIII. This produces an array of fragments ranging from 125 to 23,125 base pairs. (Actual fragment sizes are: 23130, 9416, 6557, 4361, 2322, 2027, 564, and 125 bp.) Different DNA ladders are commercially available depending on expected DNA length. The 1kb ladder with fragment ranging from about 0.5 kbp to 10 or 12 kbp, and the 100 bp ladder with fragments ranging from 100 bp to just above 1000 bp are frequently used.[5] There are also special DNA ladders for supercoiled DNA and RNA.

Commercial DNA markers are usually supplied with the loading dye included in a mix, due to the difficulty of visualizing DNA during electrophoresis. Commonly used are the dye pairs xylene cyanol and bromophenol blue; these migrate at approximately the same rate as DNA fragments 4000 and 500 base pairs in length respectively in a 1% agarose gel. Cresol red and orange G can also be used for this purpose; they migrate at approximately the same rate as DNA fragments 125 and 50 base-pairs respectively under the same conditions. These dyes often exhibit different colours depending on the pH of the buffer used during the run.

Protein markers

Protein markers can be prestained or unstained prior to loading. Commercially available prestained markers are useful since they are clearly visible as the run progresses. These are made by conjugating the proteins to colored dyes. Unstained dyes are stained during the normal visualization process of the gel, commonly through the use of coomassie blue or Flamingo.

Different Types of Molecular Markers

Many kinds of molecular markers exist, and each possesses unique characteristics. Before selection of molecular marker, it is important to become familiar with these characteristics and properties. In a particular instance one type may be more appropriate than another. Here are some examples of the more commonly used molecular markers.

RFLP

Restriction fragment length polymorphism is a technique used to detect variations in homologous DNA.[6] Specific restriction endonucleases are used to digest DNA. The RFLP molecular marker is specific to a single fragment.

SSR

Simple Sequence Repeats, or microsatellites, are short sequences of DNA base pairs.[7] This molecular marker is most frequently used for population genetic studies.

AFLP

Amplified fragment length polymorphism is a PCR base DNA fingerprinting technique. DNA is first digested with endonucleases. The restriction fragments are then ligated together.[8] A molecular marker is then generated when specific fragments are selected for amplification.

RAPD

[8] Random amplified Polymorphic DNA is a technique that is conducted the same as AFLP. The difference is that the molecular marker is generated at random.

SNP

[9]Single nucleotide polymorphism is a technique used to detect variation in a single nucleotide.

Effects of Gel Conditions

As with experimental samples, the conditions of the gel can have an impact on the molecular-weight size marker that runs alongside them. Factors such as buffers, charge/voltage, and concentration of gel can affect the mobility and/or appearance of your marker/ladder/standard. These elements need to be taken into consideration when selecting a marker and when analyzing the final results on a gel.

Buffers

DNA Electrophoresis

Buffers act to 1) establish pH, and 2) provide ions to support conductivity. In DNA electrophoresis, the TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA) are the usual buffers of choice.[10] TBE buffer preferred for small DNA pieces, whereas TAE is better suited for fragments greater than 1500 base pairs. In terms of buffering capacity, TAE is lower when compared to TBE; this generally results in slower mobility of the DNA. TBE is also capable of better resolution. However, TAE is the more preferred choice if you plan to gel extract DNA.[11]

It must be noted that water cannot act as a substitute for one of these buffers, as the DNA will not migrate along the gel.[10] Furthermore, using water instead of buffer will result in the gel melting.[12]

Protein Electrophoresis

As with DNA electrophoresis, buffers can affect the mobility of both the marker and the samples. The pH of the buffer varies with the system used and consequently, each buffer system will have a different effect of the charge of a protein or proteins.[13] In addition, in the case of SDS-PAGE, the binding affinity for SDS can be impacted by the buffering system.[13] Even when using the same percentage and type of gel, the same proteins will migrate at different rates depending on the buffer used.[13]

Charge/Voltage

DNA Electrophoresis

In terms of voltage, the recommended range is between 4 and 10 V/cm (i.e., volts/cm).[12] Agarose gels are usually run at a voltage of 5 V/cm.[10][14] It should be noted that the distance unit, cm, refers to the distance between the electrodes (i.e., the anode and the cathode) and not the length of the gel itself.[10][14]

Voltages too far below or above this range will affect the mobility and the resolution of the bands. Low voltages will decrease the mobility and will cause the bands to broaden. On the other hand, high voltages will decrease the resolution of the bands. This is largely due to the fact that voltages that are too high can cause the gel to overheat, and even melt.[12]

Protein Electrophoresis

Voltage plays a role in the mobility of proteins on a gel. Proteins will migrate faster at higher voltages. Consequently, the gel running time will be shorter. Conversely, higher voltages can result in greater band diffusion. Also, if the voltage is too high, the temperature in the electrophoresis chamber can become such that the gel begins to melt.[13]

The voltage that a gel should be run at depends on the type of the gel. For some gels, the voltage remains constant throughout the run, whereas, with other gels, the initial voltage is allowed to remain constant for a specified time before it is increased. This second voltage is then used for a specific time frame, after which, it may also be increased.[13]

Concentration

DNA Electrophoresis

Agarose concentration must be taken into account when selecting a marker. The gel percentage effects the migration of the DNA.[10][14] Generally, the higher the gel concentration, the slower the rate at which the DNA will move through the gel. This is in addition to the role molecular weight plays in the migration of a DNA ladder or sample, that is to say, that the higher the molecular weight, the slower the DNA will migrate.[10][14] Another issue to consider is the conformation of the DNA (i.e., closed circular (usually supercoiled), nicked circular, linear, etc.) Typically, supercoiled DNA will migrate the fastest whereas linear DNA will have the slowest migration rate when compared on a gel of a particular concentration.[14]

Gel concentration also has an impact on the ability to visualize the bands run out on the gel. Smaller bands are better separated resolved on a higher percentage gel, whereas increased molecular-weight bands are more easily visualized on a lower percentage gel.[10]

Protein Electrophoresis

In terms of percentage, gels used for protein electrophoresis can be broken down into single-percentage gels and gradient gels.[15] Single-percentage gels are also referred to as linear gels.[13] For linear gels, the selected percentage usually falls between 7.5% and 20% Common percentage ranges for gradient gels are 4-15% and 10-20%. Each type of gel has its own advantages. For instance, linear gels are preferred when several proteins have similar molecular weights; better separation between these proteins will be displayed by a linear gel.[15] On the other hand, gradient gels are a better choice when the samples of interest contain proteins of vastly different molecular weights or that cover a large range of molecular weights.[13][15]

References


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