Bisacrylamide crosslinker

Polyacrylamide gels are chemically formed through polymerization of the monomeric acrylamide molecule in the presence of a variable quantity of the bisacrylamide cross-linker. The polymerization process is initiated by the generation of free radicals provided by ammonium persulfate and stabilized by the compound TEMED (N,N,N',N'-tetramethylethylenediamine). This polymerization leads to the formation of long strands of acrylamide monomer with occasional cross-links provided by the bisacrylamide connector.

Polyacrylamide pore sizes can be decreased by increasing the overall concentration of acrylamide (both monomer plus cross-linker). The value of total acrylamide concentration in the gel solution is typically expressed as %T. The ratio of the monomer to the cross-linker may also be varied. The smallest pore sizes have been shown to occur when the cross-linker is 5% of the total acrylamide weight, or 5%C. A common gel solution used in STR allele separations is 5%T, 5%C. Another way this solution might be described is 5% acrylamide:bis (19:1).

One of the challenges in pouring or casting a slab gel is avoiding bubble formation. The polymerization process generates heat that can lead to bubbles forming in the gel. Gel mixtures are sometimes degassed under a vacuum for a short period of time prior to polymerization to remove any gases in the solution that might give rise to bubbles as the gel is solidifying. Sometimes bubbles occur in spite of great effort to avoid them. These gels may still be used as long as the bubbles are not in a lane where a sample will be run and/or do not interfere with the region of detection in fluorescence.

PA gels may be run in either a horizontal or a vertical format. The type of gel box defines the running format. Detection of DNA bands in polyacrylamide gels may be performed with fluorescent dyes or silver staining as will be described in Chapter 13.

NATIVE VERSUS DENATURING ELECTROPHORESIS CONDITIONS

Under normal conditions, the two complementary strands of DNA will remain together. Electrophoresis systems that perform the DNA separation while keeping the complementary strands together as double-stranded DNA are often referred to as 'native' or 'non-denaturing'. On the other hand, a separation system that possesses an environment capable of keeping the DNA strands apart as single-stranded DNA is usually referred to as a 'denaturing' system.

Generally better resolution between closely sized DNA molecules can be achieved with denaturing systems. This improved resolution is achieved because single-stranded DNA is more flexible than double-stranded DNA and therefore interacts with the sieving medium more effectively allowing closely sized molecules to be differentially separated. Additionally, natural conformation in DNA molecules, sometimes referred to as secondary structure, is eliminated in a denaturing environment.

To achieve a denaturing environment, chemicals, such as formamide and urea, may be used to keep the complementary strands of DNA apart from one another. The addition of six molar urea is a common technique for making a denaturing gel. Formamide and urea form hydrogen bonds with the DNA bases and prevent the bases from interacting with their complementary strand. The temperature of the separation or the pH of the solution may also be raised to aid in keeping the complementary strands of DNA apart.

A popular method for achieving denatured DNA strands (prior to electrophoresis) is to dilute the samples in 100% formamide. The samples are then heated to 95°C to denature the DNA strands, and then 'snap cooled' on ice by bringing them from the heated 95°C environment immediately to 0°C by placing them on ice.

PROBLEMS WITH GELS

The process of preparing a polyacrylamide gel involves a number of steps including cleaning and preparing the gel plates, combining the gel materials, pouring the gel, waiting for it to set-up, and finally removing the comb. These steps are time consuming and rather labor intensive and represent mundane tasks in the laboratory. In addition, the acrylamide gel materials are known neurotoxins and need to be handled with care.

Precast gels have also become popular due to the time and labor involved with preparing the gel plates, pouring the gel, and waiting for it to set. However, one still has to load the DNA samples very carefully into each well (to prevent contamination from adjacent wells). The development of capillary electrophoresis has excited many DNA scientists because the tedious processes of gel pouring and sample loading have been automated with this technique.

CAPILLARY ELECTROPHORESIS

Capillary electrophoresis (CE) is a relatively new addition to the electrophore-sis family. The first CE separations of DNA were performed just over a decade ago in the late 1980s. Since the introduction of new CE instrumentation in the mid-1990s, the technique has gained rapidly in popularity and for good reason. While slab-gel electrophoresis has been a proven technique for over 30 years, there are a number of advantages to analyzing DNA in a capillary format.

ADVANTAGES OF CE OVER SLAB GELS

First and foremost, the injection, separation, and detection steps can be fully automated permitting multiple samples to be run unattended. In addition, only minute quantities of sample are consumed in the injection process and samples can be easily retested if needed. This is an important advantage for precious forensic specimens that often cannot be easily replaced.

Separation in capillaries may be conducted in minutes rather than hours due to higher voltages that are permitted with improved heat dissipation from capillaries. Another advantage is that quantitative information is readily available in an electronic format following the completion of a run. No extra steps such as scanning the gel or taking a picture of it are required. Lane tracking is not necessary since the sample is contained within the capillary, nor is there fear of cross-contamination from samples leaking over from adjacent wells with CE.

DISADVANTAGES OF CE

The one major disadvantage of CE instruments is throughput. Due to the fact that samples are analyzed sequentially one at a time, single capillary instruments are not easily capable of processing high numbers of samples or sample throughputs. As will be discussed in Chapters 14 and 17, however, capillary array systems have been developed to run multiple samples in parallel and those vastly improve the sample throughput.

CE instruments require a higher start-up cost (more than $50 000) than slab-gel electrophoresis systems and this fact prohibits some laboratories from using them. Nevertheless, CE instruments are quickly becoming the principal workhorses in a number of forensic DNA typing laboratories because of their automation and ease of use.

COMPONENTS OF CE

The primary elements of a CE instrument include a narrow capillary, two buffer vials, and two electrodes connected to a high-voltage power supply. CE systems also contain a laser excitation source, a fluorescence detector, an autosampler to hold the sample tubes, and a computer to control the sample injection and detection (Figure 12.3). CE capillaries are made of fused silica (glass) and typically have an internal diameter of 50-100 |lm and a length of 25-75 cm.

The same buffers that are used in gel electrophoresis may also be used with CE. However, instead of a gel matrix through which the DNA molecules pass, a viscous polymer solution serves as the sieving medium. Larger DNA molecules are retarded more by the linear, flexible polymer chains than smaller DNA fragments, which leads to a size-based separation analogous to the DNA passing through the pores in the cross-linked polyacrylamide gels discussed above.

Prior to injecting each sample, a new gel is 'poured' by filling the capillary with a fresh aliquot of the polymer solution. The CE can be thought of as a long, skinny gel that is only wide enough for one sample at a time.

Figure 12.3 Schematic of capillary electrophoresis instruments used for DNA analysis. The capillary is a narrow glass tube approximately 50 cm long and 50^m in diameter. It is filled with a viscous polymer solution that acts much like a gel in creating a sieving environment for DNA molecules. Samples are placed into a tray and injected onto the capillary by applying a voltage to each sample sequentially. A high voltage (e.g., 15 000 volts) is applied across the capillary after the injection in order to separate the DNA fragments in a matter of minutes. Fluorescent dye-labeled products are analyzed as they pass by the detection window and are excited by a laser beam. Computerized data acquisition enables rapid analysis and digital storage of separation results.

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  • alexandra
    How to use a crosslinker for cleaning forensic lab?
    3 months ago

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