It is an old maxim of mine that when you have excluded the impossible, then whatever remains, however improbable, must be the truth.
(Sherlock Holmes, The Adventure of the Beryl Coronet)
Over the years a number of methods have been used for detecting DNA molecules following electrophoretic separation. Early techniques involved radioactive labels and autoradiography. These methods were sensitive and effective but time consuming. In addition, the use of radioisotopes was expensive due to the need for photographic films and supplies and the extensive requirements surrounding the handling and disposal of radioactive materials.
Since the late 1980s, methods such as silver staining and fluorescence techniques have gained in popularity for detecting short tandem repeat (STR) alle-les due to their low cost in the case of silver staining and their capability of automating the detection in the case of fluorescence. Table 13.1 reviews the various methods and instruments that have been used for detecting STR alleles. This chapter will focus primarily on fluorescence detection because it now dominates the forensic DNA community. Almost all commercially available STR typing kits involve the use of fluorescently labeled polymerase chain reaction (PCR) primers. However, we will briefly cover silver staining at the end of the chapter to provide what we hope will be a useful historical perspective for those who are using fluorescence detection.
Fluorescence-based detection assays are widely used in forensic laboratories due to their capabilities for multi-color analysis as well as rapid and easy-to-use formats. Fluorescence measurements involve exciting a dye molecule and then detecting the light that is emitted from the excited dye. In the application to DNA typing with STR markers, the fluorescent dye is attached to a PCR primer that is incorporated into the amplified target region of DNA. Amplified STR
Fluorescence/ABI 373 or 377
Four different color dyes are used to label PCR products; peaks are measured during electrophoresis as they pass a laser that is scanning across the gel
Four ABI dyes are used to label PCR products; capillary electrophoresis version of ABI 377; most popular method in use today among forensic labs
Fluorescence/ABI 3100 and ABI 3100 Avant
Five-dye colors available for detection with 16 capillaries or four capillaries in parallel
Gel is scanned following electrophoresis with a 532 nm laser; typically used with three different dyes and PowerPlex STR kits
Automated detection similar to the ABI 377 but with only single color capability
Decorte and Cassiman (1996)
Near-IR dyes are used to label PCR products for automated detection similar to the ABI 377
SYBR Green stain (intercalating dye) of gel following electrophoresis; gel is scanned with 488 nm laser
Morin and Smith (1995)
PCR products are labeled with an intercalating dye during CE separation for single color detection
Butler et al. (1994)
Laser scans across multiple capillaries to detect fluorescently labeled PCR products
Following electrophoresis, gel is soaked in silver nitrate solution; silver is reduced with formaldehyde to stain DNA bands
Direct blotting electrophoresis
Following run, gel bands are blotted unto a nylon membrane, fixed with UV light, and detected with digoxygenin
Berschick et al. (1993)
P32-labeled dCTP incorporated into PCR products
Hammond et al. (1994)
IR = infrared; UV = ultraviolet.
Detection methods and instruments used for analysis of STR alleles. A wide variety of fluorescence detection instrument platforms are listed.
alleles are visualized as bands on a gel or represented by peaks on an electro-pherogram. In this section, we will first discuss some of the basics surrounding fluorescence and then follow with a review of the methods used today for labeling DNA molecules, specifically the PCR products produced from STR markers.
As mentioned above, fluorescence measurements involve exciting a dye molecule and then detecting the light that is emitted from the excited dye. A molecule that is capable of fluorescence is called a fluorophore. Fluorophores come in a variety of shapes, sizes, and abilities. The ones that are primarily used in DNA labeling are dyes that fluoresce in the visible region of the spectrum, which consists of light emitted in the range of approximately 400-600 nm.
The fluorescence process is shown in Figure 13.1. In the first step, a photon (hvex) from a laser source excites a fluorophore electron from its ground energy state (S0) to an excited transition state (S'1). This electron then undergoes con-formational changes and interacts with its environment resulting in the relaxed singlet excitation state (S1). During the final step of the process, a photon (hvem) is emitted at a lower energy when the excited electron falls back to its ground state. Because energy and wavelength are inversely related to one another, the emission photon has a higher wavelength than the excitation photon.
The difference between the apex of the absorption and emission spectra is called the Stokes shift. This shift permits the use of optical filters to separate excitation light from emission light. Fluorophores have characteristic light absorption and emission patterns that are based upon their chemical structure and the environmental conditions. With careful selection and optical filters, fluoro-phores may be chosen with emission spectra that are resolvable from one another.
ex max em max ex max em max
Figure 13.1 Illustration of the fluorescence process (a) and excitation/emission spectra (b). In the first step of the fluorescence process, a photon (hvxx) from a laser source excites the fluoro-phore (dye molecule) from its ground energy state (S0) to an excited transition state (S'1). The fluoro-phore then undergoes conformational changes and interacts with its environment resulting in the relaxed singlet excitation state (S1). During the final step of the process, a photon (hvm) is emitted at a lower energy. Because energy and wavelength are inversely related to one another, the emission photon has a higher wavelength than the excitation photon.
As will be discussed later in the chapter, this capability permits the use of multiple fluorophores to measure several different DNA molecules simultaneously. The rate at which samples can be processed is much greater with multiple fluorophores than measurements involving a single fluorophore.
There are a number of factors that affect how well a fluorophore will emit light, or fluoresce. These factors include the following (Singer and Johnson 1997):
■ Molar extinction coefficient: the ability of a dye to absorb light;
■ Quantum yield: the efficiency with which the excited fluorophore converts absorbed light to emitted light;
■ Photo stability: the ability of a dye to undergo repeated cycles of excitation and emission without being destroyed in the excited state, or experiencing 'photobleaching';
■ Dye environment: factors that affect fluorescent yield include pH, temperature, solvent, and the presence of quenchers, such as hemoglobin.
The overall fluorescence efficiency of a dye molecule depends on a combination of these four factors. For example, fluorescein dyes have a lower molar extinction coefficient than rhodamine dyes yet the fluorescein dyes fluoresce well because they have higher quantum yields. Thus, fluorescein dyes do not absorb light as well but do a better job of converting the absorbed light into emitted light. This fact points out that the brightness of a fluorophore is proportional to the product of the molar extinction coefficient and the quantum yield.
SELECTING THE OPTIMAL FLUOROPHORE FOR AN APPLICATION
Optimal dye selection requires consideration of the spectral properties of fluorescent labels in relation to the characteristics of the instrument used for detection (Singer and Johnson 1997). The intensity of the light emitted by a fluorophore is directly dependent on the amount of light that the dye has absorbed. Thus, the excitation source is very important in the behavior of a flu-orophore. Other important instrument parameters to be considered include optical filters used for signal discrimination and the sensitivity and spectral response of the detector.
Lasers are an effective excitation source because the light they emit is very intense and at primarily one wavelength. One of two different lasers is typically used to excite fluorescent dyes in the visible spectrum. The argon ion gas laser (Ar+) produces light at 488 nm and 514.5 nm (see Chapter 14). This laser is by far the most popular for applications involving fluorescent DNA labeling because a number of dyes are available that closely match its excitation capabilities. The other laser that is used is the solid-state Nd:YAG laser that produces a beam of light at 532 nm (see Chapter 14).
A significant advantage of fluorescent labeling over other methods is the ability to record two or more fluorophores separately using optical filters and a flu-orophore separation algorithm known as a matrix. With this multi-color capability, components of complex mixtures can be labeled individually and identified separately in the same sample. Fluorescent signals are differentiated by using filters that block out light from adjacent regions of the spectrum. Signal discrimination by software matrix deconvolution of the various dye colors will be discussed later in this chapter.
A fluorescence detector is a photosensitive device that measures the light intensity emitted from a fluorophore. Detection of low-intensity light may be accomplished with a photomultiplier tube (PMT) or a chargecoupled device (CCD). In both cases, the action of a photon striking the detector is converted to an electric signal. The strength of the resultant current is proportional to the intensity of the incident light. This light intensity is typically reported in arbitrary units, such as relative fluorescence units (RFUs).
Fluorescent labeling of PCR products may be accomplished in one of three ways: (1) incorporating a fluorescent dye into the amplicon through a 5'-end labeled oligonucleotide primer; (2) incorporating fluorescently labeled deoxynucleotides (dNTPs) into the PCR product; and (3) using a fluorescent intercalating dye to bind to the DNA (Mansfield and Kronick 1993). These three methods are illustrated in Figure 13.2.
Each method of labeling DNA has advantages and disadvantages. Intercalating dyes may be used following PCR and are less expensive than the other two methods. However, they can only be used to analyze DNA fragments in a single color, which means that all of the molecules must be able to be separated in terms of size. On the other hand, dye labeled primers are popular because only a single strand of a PCR product is labeled, which simplifies data interpretation because the complementary DNA strand is not visible to the detector. Dye-labeled primers also enable multiple amplicons to be labeled simultaneously in an independent fashion.
The addition of a fluorescent dye to a DNA fragment impacts the DNA molecule's electrophoretic mobility. This is because the physical size and shape of the dye changes the overall size of the dye-DNA conjugate. The ionic charge, which is present on the dye, also alters the charge to size ratio of the nucleic acid conjugate. Fluorescent dyes that are covalently coupled to STR primers slightly alter the electrophoretic mobility of a STR allele PCR product moving through a gel or capillary. However, software corrections are used to mitigate this problem. In addition, genotyping of alleles is always performed relative to
Methods for fluorescently labeling DNA fragments. Double-stranded DNA molecules may be labeled with fluorescent intercalating dyes (a). The fluorescence of these dyes is enhanced upon insertion between the DNA bases. Alternatively a fluorescent dye may be attached to a nucleotide triphosphate and incorporated into the extended strands of a PCR product (b). The most common method of detecting STR alleles is the use of fluorescent dye labeled primers (c). These primers are incorporated into the PCR product to fluorescently label one of the strands.
(a) Unlabeled DNA
Intercalator inserts between base pairs on double-stranded DNA
Intercalator inserts between base pairs on double-stranded DNA
DNA labeled with intercalating dye
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