Protocols and Applications Guide (US Letter Size) - Promega

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|||||||||||||| 14DNA-Based Human Identification GIN018 Automation in a forensic laboratory (www.promega.com /profiles/702/702_12.html) GIN013 Robotic extraction of mock sexual assault samples using the Biomek® 2000 and the DNA IQ System (www.promega.com /profiles/501/501_03.html) AN102 Extraction and isolation of DNA from blood cards and buccal swabs in a 96-well format (www.promega.com /appnotes/an102/an102.html) GE181 Validation Guide for the DNA IQ Reference Sample Kit for Maxwell® 16. (www.promega.com /maxwell16/maxwell_dna_iq_reference_manual.pdf) D. Automated Differential Extraction The Automated Differex System allows high-throughput, automated differential extraction of sexual assault samples and integrates with the DNA IQ System for subsequent DNA purification. Up to 48 samples can be processed in less than 5 hours using the automated Differex protocol; 40 samples can be processed if wells are reserved for DNA standards in downstream DNA quantitation. Two independent, but related, automated methods are used to process differential extractions: the automated Differex method and automated DNA IQ for Differex method. The automated Differex method involves separating samples into epithelial and sperm fractions, while the automated DNA IQ for Differex method purifies genomic DNA from each fraction. The protocol begins with a proteinase K digestion of swab samples in a Slicprep 96 Device, followed by centrifugation to pellet the sperm and separate digestion products from the solid support material. The plate containing the sperm pellets and supernatants is placed onto the deck of a robotic workstation, and the automated Differex method begins. Automated Differex protocols exist for the Biomek ® 2000 and Biomek ® 3000 workstations and Tecan Freedom EVO ® 100 liquid-handler. The automated Differex method combines the Differex System reagents and DNA IQ Resin in a novel pellet-capping process. First, the robot dispenses DNA IQ Resin directly on top of each sperm pellet. Upon application of a magnetic field, the paramagnetic resin particles form a capping layer, which allows supernatant manipulation without pellet disruption (Figure 14.3). A portion of the supernatant is removed from each sample well and moved to an adjacent well for downstream processing as the epithelial fraction. The remaining supernatant is removed from the pellet and discarded or, if desired, archived. The resin-capped sperm pellets are washed four times to dilute and remove any residual epithelial DNA-containing supernatant. During the third wash, the resin-capped sperm pellets are resuspended to release any trapped epithelial Protocols & Applications Guide www.promega.com rev. 6/09 material. Resuspending the pellets requires a manual step to centrifuge the sample plate and pellet the sperm. Following this centrifugation the robot adds a second aliquot of DNA IQ Resin to re-establish the pellet cap. The Differex Separation Solution is used to float the epithelial DNA-containing wash buffer away from the capped pellet to further improve epithelial DNA removal. The fourth and final wash is performed to complete the automated Differex extraction. The sperm and epithelial fractions then undergo automated DNA isolation using the DNA IQ System. Integration of DNA IQ and Differex System components results in higher sperm fraction yields and reproducible, automated sexual assault sample processing. Figure 14.3. The Automated Differex System. The automated Differex method uses DNA IQ Resin to cap the sperm pellet after differential lysis and centrifugation. The MagnaBot® Flat Top Magnetic Separation Device immobilizes the DNA IQ Resin, holding the sperm pellet in place during automated wash steps. The Separation Solution helps remove any remaining digestion buffer and epithelial DNA from the sperm pellet. Additional Resources for Automated Differex System Technical Bulletins and Manuals EP030 Automated Differex System Protocol for the Tecan Freedom EVO® System (www.promega.com /tbs/ep030/ep030.html) EP031 Automated Differex System Protocol for the Beckman Coulter Biomek® 2000 (www.promega.com /tbs/ep031/ep031.html) EP032 Automated Differex System Protocol for the Beckman Coulter Biomek® 3000 (www.promega.com /tbs/ep032/ep032.html) PROTOCOLS & APPLICATIONS GUIDE 14-11
|||||||||||||| 14DNA-Based Human Identification Promega Publications GIN024 Automating the Differex System. (www.promega.com /profiles/1002/1002_19.html) E. Automated DNA Normalization and PCR Setup Promega has worked in conjunction with Beckman Coulter to develop a tool for DNA normalization and PCR setup on the Biomek® 2000 workstation. The Genetic Identity version of the Normalization Wizard, available from Beckman Coulter, dilutes DNA samples at different concentrations to the desired final concentration, then assembles the amplification reactions. The robot transfers PCR master mix to strip tubes or plates, then adds a fixed volume of the normalized DNA. The samples are capped manually and placed directly in a thermal cycler for amplification. While this automation section focuses on the Beckman Coulter Biomek® 2000 and 3000 laboratory automation workstations and Tecan Freedom EVO® liquid handlers, automation users and other instrument suppliers have developed their own methods for human DNA quantitation, normalization and PCR setup using other instruments (www.promega.com /applications/hmnid/automation/automation_sp.htm). Though these methods have not been validated by Promega scientists, they might be of interest to those involved in automation. For example, PerkinElmer has developed methods for DNA quantitation, subsequent normalization and PCR setup on the JANUS® Automated Workstation (www.promega.com /applications/hmnid/automation/automation_qnp.htm). Additional Resources for DNA Normalization and PCR Setp Promega Publications GIN018 Automated DNA normalization and STR multiplex setup methods. (www.promega.com /profiles/702/702_11.html) VII. References Anderson, S. et al. (1981) Sequence and organization of the human mitochondrial genome. Nature 290, 457–65. Budowle, B. et al. (1991) Analysis of the VNTR locus D1S80 by the PCR followed by high-resolution PAGE. Am. J. Hum. Genet. 48, 137–44. Butler, J.M. (2005) Forensic DNA Typing, Elsevier Academic Press, Burlington, MA. Edwards, A. et al. (1991a) DNA typing with trimeric and tetrameric tandem repeats: Polymorphic loci, detection systems, and population genetics. The Second International Symposium on Human Identification 1991, Promega Corporation , 31–52. Protocols & Applications Guide www.promega.com rev. 6/09 Edwards, A. et al. (1991b) DNA typing and genetic mapping with trimeric and tetrameric tandem repeats. Am. J. Hum. Genet. 49, 746–56. Edwards, A. et al. (1992) Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups. Genomics 12, 241–53. Ensenberger, M.G. and Fulmer, P.M. (2009) The PowerPlex® 16 HS System. Profiles in DNA 12(1), 9–11. Gill, P. (2001) An assessment of the utility of single nucleotide polymorphisms (SNPs) for forensic purposes. Int. J. Leg. Med. 114, 204–10. Gill, P., Jeffreys, A.J. and Werrett, D.J. (1985) Forensic application of DNA ‘fingerprints’. Nature 318, 577–9. Gusmão, L. and Carracedo, A. (2003) Y chromosome-specific STRs. Profiles in DNA 6(1), 3–5. Holland, P.M. et al. (1991) Detection of specific polymerase chain reaction product by utilizing the 5′→3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. USA 88, 7276–80. Jeffreys, A.J., Wilson, V. and Thein, S.L. (1985a) Hypervariable 'minisatellite' regions in human DNA. Nature 314, 67–73. Jeffreys, A.J., Wilson, V. and Thein, S.L. (1985b) Individual-specific 'fingerprints' of human DNA. Nature 316, 76–9. Krenke, B.E. et al. (2002) Validation of a 16-locus fluorescent multiplex system. J. Forensic Sci. 47, 773–85. Krenke, B.E. et al. (2005) Validation of a male-specific, 12-locus fluorescent short tandem repeat (STR) multiplex. Forensic Sci. Int. 148, 1–14. Linch, C.A., Smith, S.L. and Prahlow, J.A. (1998) Evaluation of the human hair root for DNA typing subsequent to microscopic comparison. J. Forensic Sci. 43, 305–14. McLaren, R. (2007) PowerPlex® 16 versus Identifiler® Systems: Sensitivity and effects of inhibitors. Application Notes 156 , Promega Corporation. Nakamura, Y. et al. (1987) Variable number of tandem repeat (VNTR) markers for human gene mapping. Science 235, 1616–22. Phillips, C. et al. (2003) Selecting single nucleotide polymorphisms for forensic applications. Int. Congr. Ser. 1261, 18–20. Saiki, R. et al. (1985) Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350–4. Tereba, A. et al. (2004) A new, rapid method to separate sperm and epithelial cells. Profiles in DNA 7(2), 8–10. Tillmar, A.O. et al. (2008) Analysis of linkage and linkage disequilibrium for eight X-STR markers. For. Sci. Int. Genet. 3, 37–41. Warne, D. et al. (1991) Tetranucleotide repeat polymorphism at the human ß-actin related pseudogene 2 (actbp2) detected using the polymerase chain reaction. Nucleic Acid Res. 19, 6980. Wyman, A.R. and White, R. (1980) A highly polymorphic locus in human DNA. Proc. Natl. Acad. Sci. USA 77, 6754–8. PROTOCOLS & APPLICATIONS GUIDE 14-12
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CONTENTS I. Nucleic Acid Amplificat
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| 1Nucleic Acid Amplification CONTE
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| 1Nucleic Acid Amplification Unamp
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| 1Nucleic Acid Amplification Capoz
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| 1Nucleic Acid Amplification is co
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| 1Nucleic Acid Amplification One i
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| 1Nucleic Acid Amplification React
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| 1Nucleic Acid Amplification comig
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| 1Nucleic Acid Amplification inclu
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| 1Nucleic Acid Amplification polym
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| 1Nucleic Acid Amplification Addit
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| 1Nucleic Acid Amplification 3. Pl
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| 1Nucleic Acid Amplification 13. S
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| 1Nucleic Acid Amplification IX. R
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| 1Nucleic Acid Amplification Hoppe
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|| 2RNA Interference CONTENTS I. RN
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|| 2RNA Interference zebrafish (War
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|| 2RNA Interference Additional Res
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|| 2RNA Interference 2. Combine sep
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|| 2RNA Interference Target Site Se
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|| 2RNA Interference Transient Tran
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|| 2RNA Interference VII. Reference
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|| 2RNA Interference Wianny, F. and
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||| 3Apoptosis I. Introduction A. C
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||| 3Apoptosis ER stress pathway (H
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|||| 4Cell Viability CONTENTS I. In
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||||| 5Protein Expression I. Introd
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||||||| 7Cell Signaling CONTENTS I.
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||||||| 7Cell Signaling (continued
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Table 8.1. pGL4 Luciferase Reporter
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||||||||| 9DNA Purification I. Intr
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|||||||||| 10Cell Imaging I. Introd
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