LLJlil

5000

4000

3000

2000

1000-

Figure 13.7 STR Data from ABI Prism 310 Genetic Analyzer. This sample was amplified with the AmpFlSTR SGM Plus kit. Raw data prior to color separation (a) compared with GeneScan 3.1 color separated allele peaks (b). The red-labeled peaks are from the internal sizing standard GS500-ROX.

because of off-scale peaks. The most common occurrence of pull-up involves small green peaks showing up under blue peaks that are off-scale. This occurs because of the significant overlap of the blue and green dyes seen in Figure 13.4. Samples can be diluted and analyzed again to reduce or eliminate the offending pull-up peak(s).

Raw data from a fluorescently labeled DNA sample is compared to the color separated processed data in Figure 13.7. DNA fragments labeled with the yellow dye NED are shown in black. Multi-component analysis is performed automatically with the GeneScan® Analysis Software using a mathematical matrix calculation.

FLUORESCENCE DETECTION PLATFORMS

As seen in Table 13.1, a number of fluorescence detection platforms exist and have been used for STR allele determination. The most popular detection platforms today for STR analysis in the United States are the ABI Prism 310 Genetic Analyzer, the ABI 3100 multi-capillary system, and the FMBIO II gel scanner. These three instruments will be reviewed more extensively in Chapter 14. The ABI 310 and FMBIO II instruments have fundamental differences in their approach to detecting fluorescently labeled PCR products. In addition, specific STR kits have been designed for using each approach.

With the ABI 310 approach, detection is performed during electrophoresis (Figure 13.8). Other instruments listed in Table 13.1 that use a similar detection format as the ABI 310 include the ABI 377, ABI 3100, ABI 3700, the ALF DNA sequencer, and the LICOR systems. Applied Biosystems has prepared the Profiler Plus™ and COfiler™ STR kits to work on any of the ABI detection platforms (i.e., ABI Prism 310, 377, 3100, and 3700). These two kits cover the 13 core CODIS STR loci with three markers in common between the sets for concordance purposes (Figure 13.9). The PowerPlex® 16 and Identifiler™ STR kits now enable amplification of all 13 CODIS loci plus two additional polymorphic STRs in a single reaction (see Figures 5.4 and 5.5).

Figure 13.8

Schematic illustration of the separation and detection of STR alleles with an ABI Prism 310 Genetic Analyzer.

Size

Sample Separation

Sample Injection

Sample Separation

H

k

H

H

Capillary

Argon ion LASER

ABI Prism spectrograph

ABI Prism spectrograph

Color Separation

Fluorescence

Fluorescence

Capillary

Color Separation

CCD Panel (with virtual filters)

Sample Detection

Mixture of dye-labeled PCR products from multiplex PCR reaction

Processing with GeneScan/Genotyper software

Processing with GeneScan/Genotyper software

Sample Interpretation

Sample Interpretation

Profiler Plus™ multiplex STR result

DNA size(bp)

150 175 200 225 250 275 300

D3S1358

Amel

D21S11

FGA D13S317

D7S820

D18S51

D18S51

COfiler™ multiplex STR result

150 175 200 225 250

DNA size(bp) 275 300

3200 2400 1600 800 0

D3S1358

TH01

Amel

TPOX

D7S820

CSF1PO

D16S539

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

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

Applied Biosystems (1998) AmpFlSTR Profiler Plus™ PCR Amplification Kit User's Manual. Foster City, California: Applied Biosystems.

Bassam, B.J., Caetano-Anolles, G. and Gresshoff, P.M. (1991) Analytical Biochemistry, 196, 80-83.

Berschick, P., Henke, L. and Henke, J. (1993) Proceedings of the Fourth International Symposium on Human Identification, pp. 201-204. Madison, Wisconsin: Promega Corporation.

Budowle, B., Baechtel, F.S., Comey, C.T., Giusti, A.M. and Klevan, L. (1995) Electrophoresis, 16, 1559-1567.

Buel, E., Schwartz, M. and LaFountain, M.J. (1998) Journal of Forensic Sciences, 43, 164-170.

Butler, J.M., McCord, B.R., Jung, J.M. and Allen, R.O. (1994) BioTechniques, 17, 1062-1070.

Butler, J.M., Buel, E., Crivellente, F. and McCord, B.R. (2004) Electrophoresis, 25, 1397-1412.

Decorte, R. and Cassiman, J.-J. (1996) Electrophoresis, 17, 1542-1549.

Edwards, A., Civitello, A., Hammond, H.A. and Caskey, C.T. (1991) American Journal of Human Genetics, 49, 746-756.

Frazier, R.R.E., Millican, E.S., Watson, S.K., Oldroyd, N.J., Sparkes, R.L., Taylor, K.M., Panchal, S., Bark, L., Kimpton, C.P. and Gill, P.D. (1996) Electrophoresis, 17, 1550-1552.

Greenspoon, S.A., Ban, J.D., Pablo, L., Crouse, C.A., Kist, F.G., Tomsey, C.S., Glessner, A.L., Mihalacki, L.R., Long, T.M., Heidebrecht, B.J., Braunstein, C.A., Freeman, D.A., Soberalski, C., Nathan, B., Amin, A.S., Douglas, E.K. and Schümm, J.W. (2004) Journal of Forensic Sciences, 49, 71-80.

Hammond, H.A., Jin, L., Zhong, Y., Caskey, C.T. and Chakraborty, R. (1994) American Journal of Human Genetics, 55, 175-189.

Huang, N.E., Schümm, J.W. and Budowle, B. (1995) Forensic Science International, 71, 131-136.

Lazaruk, K., Walsh, P.S., Oaks, F., Gilbert, D., Rosenblum, B.B., Menchen, S., Scheibler, D., Wenz, H.M., Holt, C. and Wallin, J. (1998) Electrophoresis, 19, 86-93.

Lee, S.B., Buoncristiani, M., Schumm, J.W. and Wingeleth, D. (1995) Proceedings of the Fifth International Symposium on Human Identification, pp. 104-111. Madison, Wisconsin: Promega Corporation.

Lins, A.M., Micka, K.A., Sprecher, C.J., Taylor, J.A., Bacher, J.W., Rabbach, D., Bever, R.A., Creacy, S. and Schumm, J.W. (1998) Journal of Forensic Sciences, 43, 1178-1190.

Mansfield, E.S. and Kronick, M.N. (1993) BioTechniques, 15, 274-279.

Mansfield, E.S., Robertson, J.M., Vainer, M., Isenberg, A.R., Frazier, R.R., Ferguson, K., Chow, S., Harris, D.W., Barker, D.L., Gill, P.D., Budowle, B. and McCord, B.R. (1998) Electrophoresis, 19, 101-107.

Merril, C.R., Washart, K.M. and Allen, R.C. (1998) In Tietz, D. (ed) Nucleic Acid Electrophoresis. New York: Springer.

Micka, K.A., Sprecher, C.J., Lins, A.M., Comey, C.T., Koons, B.W., Crouse, C., Endean, D., Pirelli, K., Lee, S.B., Duda, N., Ma, M. and Schumm, J.W. (1996) Journal of Forensic Sciences, 41, 582-590.

Morin, P.A. and Smith, D.G. (1995) BioTechniques, 19, 223-227.

Roy, R., Steffens, D.L., Gartside, B., Jang, G.Y. and Brumbaugh, J.A. (1996) Journal of Forensic Sciences, 41, 418-424.

Schumm, J.W., Lins, A.M., Sprecher, C.J. and Micka, K.A. (1995) Proceedings from the Sixth International Symposium on Human Identification, pp. 10-19. Madison, Wisconsin: Promega Corporation.

Sgueglia, J. B., Geiger, S. and Davis, J. (2003) Analytical and Bioanalytical Chemistry, 376, 1247-1254.

Singer, V.L. and Johnson, I.D. (1997) Proceedings of the Eighth International Symposium on Human Identification, pp.70-77. Madison, Wisconsin: Promega Corporation.

Wang, Y., Ju, J., Carpenter, B.A., Atherton, J.M., Sensabaugh, G.F. and Mathies, R.A. (1995) Analytical Chemistry, 67, 1197-1203.

Worley, J.M., Ma, M., Lee, S.B., Lins, A.M., Schumm, J.W. and Mansfield, E.S. (1994) Proceedings from the Fifth International Symposium on Human Identification, pp. 109-117. Madison, Wisconsin: Promega Corporation.

CHAPTER 14

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This book discusses the futility of curing stammering by common means. It traces various attempts at curing stammering in the past and how wasteful these attempt were, until he discovered a simple program to cure it. The book presents the life of Benjamin Nathaniel Bogue and his struggles with the handicap. Bogue devotes a great deal of text to explain the handicap of stammering, its effects on the body and psychology of the sufferer, and its cure.

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