It is a capital mistake to theorize before one has data. Insensibly one begins to twist facts to suit theories, instead of theories to suit facts.
(Sherlock Holmes, A Scandal in Bohemia)
A polymerase chain reaction (PCR) reaction in which short tandem repeat (STR) alleles are amplified produces a mixture of DNA molecules that present a challenging separation problem. A multiplex PCR can produce 20 or more DNA fragments that must be resolved from one another. In addition, single base resolution is required to distinguish between closely spaced alleles (e.g., TH01 alleles 9.3 and 10). The typical separation size range where this single base resolution is needed is between 100 and 400 bp. Additionally, it is important that the separation method be reproducible and yield results that can be compared among laboratories.
In order to distinguish the various molecules from one another, a separation step is required to pull the different sized fragments apart. The separation is typically performed by a process known as electrophoresis and is either conducted in a slab-gel or capillary environment. This chapter will discuss the theory and background information on separation methods. Chapter 13 will cover how the bands in gel electrophoresis and the peaks in capillary elec-trophoresis are actually generated and detected. Chapter 14 will discuss specific techniques utilizing capillary electrophoresis and slab-gel electrophoresis that are widely used by forensic DNA typing laboratories.
PCR products from short tandem repeat DNA must be separated in a fashion that allows each allele to be distinguished from other alleles. Heterozygous alleles are resolved in this manner with a sized based separation method known as elec-trophoresis. The separation medium may be in the form of a slab gel or a capillary.
The word 'electrophoresis' comes from the Greek electron (charge) and the Latin phore (bearer). Thus, the process of electrophoresis refers to electrical charges carried by the molecules. In the case of DNA, the phosphate groups on the backbone of the DNA molecule have a negative charge. Nucleic acids are acids because the phosphate groups readily give up their H+ ions making them negatively charged in most buffer systems. Under the influence of an electric field, DNA molecules will migrate away from the negative electrode, known as the cathode, and move towards the positive electrode, known as the anode. The higher the voltage, the greater the force felt by the DNA molecules and the faster the DNA moves.
The movement of ions in an electric field generates heat. This heat must be dissipated or it will be absorbed by the system. Excessive heat can cause a gel to generate bands that 'smile' or in very severe cases the gel can literally melt and fall apart. As will be described at the end of the chapter, performing electrophoresis in a capillary is an advantage because heat can be more easily dissipated from the capillary, which has a high surface area-to-volume ratio.
Schematic of a gel electrophoresis system. The horizontal gel is submerged in a tank full of electrophoresis buffer. DNA samples are loaded into wells across the top of the gel. These wells are created by a 'comb' placed in the gel while it is forming. When the voltage is applied across the two electrodes, the DNA molecules move towards the anode and separate by size. The number of lanes available on a gel is dependent on the number of teeth in the comb used to define the loading wells. At least one lane on each gel is taken up by a molecular weight size standard that is used to estimate the sizes of the sample bands in the other lanes.
Slab gels consist of a solid matrix with a series of pores and a buffer solution through which the DNA molecules pass during electrophoresis. Gel materials are mixed together and poured into a mold to define the structure of the slab gel. A sample 'comb' is placed into the gel such that the teeth of the comb are imbedded in the gel matrix. After the gel has solidified, the comb is removed leaving behind wells that are used for loading the DNA samples. The basic format for a gel electrophoresis system is shown in Figure 12.1.
Two types of gels are commonly used in molecular biology and forensic DNA laboratories today to achieve DNA separations. Agarose gels have fairly large pore sizes and are used for separating larger DNA molecules while polyacryl-amide gels are used to obtain high-resolution separations for smaller DNA molecules, usually below 500 or 1000 bp.
Forensic DNA typing methods use both types of gels. Restriction fragment length polymorphism (RFLP) methods use agarose gels to separate DNA fragments ranging in size from ~600 bp to ~23 000 bp. Low molecular weight DNA molecules are not well separated with agarose slab gels. On the other hand, PCR-amplified STR alleles, which range in size from ~100bp to ~400bp, are better served by polyacrylamide gels. In the case of some STR loci that contain microvari-ants, the high-resolution capability of polyacrylamide gels is essential for separating closely sized DNA molecules that may only differ by a single nucleotide.
Agarose is basically a form of seaweed and contains pores that are on the order of 2000 angstroms (A) (200 nm) in diameter. Agarose gels are easily prepared by weighing out a desired amount of agarose powder and mixing it with the electrophoresis buffer. This mixture can be quickly brought to a boil by microwaving the solution whereupon the agarose powder goes into solution. After the solution cools down slightly, it is poured into a gel box to define the gel shape and thickness.
A comb is added to the liquid agarose before it cools to form wells with its teeth in the jelly-like substance that results after the gel 'sets.' Once the agarose has gelled the comb is removed leaving behind little wells that can hold 5-10 ||L of sample or more depending on the size of the teeth and the depth at which they were placed in the agarose gel. The comb teeth define the number of samples that can be loaded onto the gel as well as where the lanes will be located. There are predominantly two types of combs: square-tooth and sharks-tooth.
Electrophoresis buffer is poured over the gel until it is fully submerged. Two buffers are commonly used with electrophoresis, Tris-acetate-EDTA (TAE) and Tris-borate-EDTA (TBE). Samples are mixed with a loading dye and carefully pipetted into each well of the submerged gel. This loading dye contains a mixture of bromophenol blue, a dark blue dye which helps to visually see the sample, and sucrose to increase the sample's viscosity and help it stay in the well prior to turning on the voltage and initiating electrophoresis.
The number of samples that can be run in parallel on the gel are defined by the number of teeth on the comb added to the gel before it sets (and hence the number of wells that will be created). Typically between eight and 24 samples
Illustration of polyacry-lamide gel polymerization. The pore size of a gel is controlled by its degree of cross-linking, which depends on the proportions of acrylamide and bisacrylamide in the gel. Polymerization is typically induced by free radicals resulting from the chemical decomposition of ammonium persulfate. TEMED, a free radical stabilizer, is also added to the gel mixture to stabilize the polymerization process. A polyacrylamide gel is poured between two glass plates that define its dimensions. A spacer is placed between the plates to define the thickness of the gel. A typical gel size is 17 cm X 43 cm X 0.4 cm. DNA molecules flow through the polyacry-lamide gel matrix along the z-axis going into the page, much like flowing through a chain link fence with various sized holes.
are run at a time on an agarose gel. Molecular weight standards are run in some of the lanes in order to estimate the size of each DNA sample following electrophoresis.
After the samples are loaded, a cover is placed over the gel box containing the submerged gel and the electrodes on either end of the gel are plugged into a power source. The anode (positive electrode) is placed on the end of the gel furthest from the wells to draw the DNA molecules through the gel material. Typically 100-600 volts (V) are placed across agarose gels that are 10-40 cm in length, creating electric field strengths of approximately 1-10 V/cm.
As the DNA molecules are drawn through the gel, they are separated by size, the smaller ones moving more quickly and easily through the gel pores. It might help to think of the DNA molecules as marathon runners with different abilities. They all start together at the beginning and then separate during the 'race' through the gel. The smaller DNA molecules move more quickly than the larger ones through the obstacles along the gel 'race course' and thus are further along when the voltage is turned off and the 'race' completed. When the separation is completed, the gel is scanned or photographed to record the results for examination and comparison.
Polyacrylamide (PA) gels have much smaller pore sizes (~100-200Â) than agarose gels (~1500-2000Â). The average pore size of a gel is an important factor in determining the ability of a slab gel to resolve two similarly sized DNA fragments. The pores in PA gels are chemically created with a cross-linking process involving acrylamide and bisacrylamide (Figure 12.2).
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