COfiler™ multiplex STR result
150 175 200 225 250
DNA size(bp) 275 300
3200 2400 1600 800 0
Figure 13.9 AmpFlSTR® Profiler Plus™ and COfiler™ STR Data Collected on an ABI310 Capillary Electrophoresis System. The STR loci that are surrounded by a box are common to both multiplex mixes and are therefore useful as a quality assurance measure to demonstrate sample concordance.
With the FMBIO II or other gel scanning systems, detection is performed following electrophoresis (Figure 13.10). Thus, many gels can be run in separate gel rigs and detected via rapid scanning on a single FMBIO fluorescence imaging system. The Promega Corporation has created several multiplex STR kits to work with the FMBIO II detection platform: PowerPlex® 1.1, PowerPlex® 2.1, and PowerPlex® 16 BIO (Figure 13.11). These kits enable amplification of the 13 core CODIS STR loci with either the combination of PowerPlex® 1.1 and 2.1 or PowerPlex® 16 BIO, which amplifies all 13 CODIS loci plus Penta D and Penta E in a single multiplex reaction (Greenspoon et al. 2004).
These two separation and detection approaches have differing abilities to separate STR alleles of various size ranges. Every DNA fragment travels the same distance when the detector is at a fixed point relative to the injection of the sample, as with the ABI 310. On the other hand, when separation and detection are separate steps, as with the FMBIO gel scanner, DNA fragments of different sizes travel different distances through the gel. Smaller molecular weight PCR products (e.g., VWA) travel further through the gel and are thus better resolved from one another compared to the higher molecular weight species (e.g., FGA) that only move a short distance through the gel before the electrophoresis is stopped and the gel is scanned.
Figure 13.10 Schematic of gel separation and FMBIO II detection of STR alleles.
Figure 13.10 Schematic of gel separation and FMBIO II detection of STR alleles.
Silver staining of polyacrylamide gels has been useful for detecting small amounts of proteins and visualizing nucleic acids. Although not as commonly used today, silver staining procedures were used for the first commercially available STR kits from the Promega Corporation. Promega still supports silver-stain gel users although most of their customer base now uses fluorescent STR systems. Silver-stain detection methods are still quite effective for laboratories that want to perform DNA typing for a much smaller start-up cost. No expensive
Figure 13.11 PowerPlex® 16 BIO data collected on a Hitachi FMBIOIII plus Fluorescence Imaging System. Color-separated data for this same gel is contained in Figure 14.9. Figure courtesy of Margaret Kline, NIST.
instruments are needed, simply a gel box for electrophoresis and some silver nitrate and other developing chemicals.
The procedure for silver staining is performed by transferring the gel between pans filled with various solutions that expose the DNA bands to a series of chemicals for staining purposes (Bassam et al. 1991). First, the gel is submerged in a pan of 0.2% silver nitrate solution. The silver binds to the DNA and is reduced with formaldehyde to form a deposit of metallic silver on the DNA molecules in the gel. A photograph is then taken of the gel to capture images of the silver-stained DNA strands and to maintain a permanent record of the gel. Alternatively, the gels themselves may be sealed and preserved.
ADVANTAGES AND DISADVANTAGES OF SILVER STAINING
Silver staining is less hazardous than radioactive detection methods although not as convenient as fluorescence methods. Most reagents for silver staining are harmless and thus require no special precautions for handling. The primary advantage of silver staining is that the technique is inexpensive. The developing chemicals are readily available at low cost. The PCR products do not need any special labels, such as fluorescent dyes. The staining may be completed within half an hour and with a minimal number of steps. Sensitivity is approximately 100 times higher than that obtained with ethidium bromide staining (Merril et al. 1998). However, a major disadvantage to data interpretation is that both DNA strands may be detected in a denaturing environment leading to two bands for each allele. In addition, only one 'color' exists, which makes PCR product size differences the only method for multiplexing STR markers.
REFERENCES AND ADDITIONAL READING
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