By Angel Carracedo
A cutting-edge selection of quite simply reproducible laboratory tools for DNA identification research, together with Y chromosome haplotyping, mtDNA, and SNP typing. The e-book deals well-tested protocols for DNA quantification utilizing real-time PCR on forensic samples and for the decision of the variety of amelogenine gene copies. For forensic geneticists, there are with no trouble reproducible equipment for species id, old DNA, and pharmacogenetics. extra chapters handle new functions within the forensic genetics lab, one of these species identity or typing of CYP polymorphisms for the research of difficult to medications.
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Additional info for Forensic DNA Typing Protocols (Methods in Molecular Biology)
Gibson, U. , Heid, C. , and Williams, P. M. (1996). A novel method for real 15 time quantitative RT-PCR. Genome Res. 6, 995–1001. 16. , and Watson, R. (1993). Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology 11, 1026–1030. 17. , and Saiki, R. K. (1988). A general method of in vitro 17 preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 16, 7351–7367. 18. Ho, S. , Hunt, H. , Horton, R. , Pullen, J.
18. 0, 10 mM CaCl 2, 100 mM sodium chloride, 2% sodium dodecyl sulfate. 19. Sterile Petri dishes. 5-mL Sarstedt tubes with screw caps. 2. rtPCR Quantitation 1. ExoSapIT Amersham/Pharmacia. 2. TOPO TA Cloning Kit, Invitrogen Life Technologies. 3. TaqMan® Universal PCR Master Mix, No AmpErase® UNG. 3. Sequence of Primers and Fluorogenic Probes Nuclear Target: RB1 Gene Primers for cloning: pRB1 F pRB1 R 5'-AGGTTGCTAACTATGAAACACTGGC-3' 5'-CCATCTCAGCTACTGGAAAACATTC-3' Primers for 79-bp amplicon: (5) RB1-2672 F 5'-CCAGAAAATAAATCAGATGGTATGTAACA-3' RB1-2750 R 5'-TGGTTTAGGAGGGTTGCTTCC-3' Primers for 156-bp amplicon: pRB1 F RB1-2750 R 5'-AGGTTGCTAACTATGAAACACTGGC-3' 5'-TGGTTTAGGAGGGTTGCTTCC-3' Primers for 246-bp amplicon: pRB1 F pRB1 R 5'-AGGTTGCTAACTATGAAACACTGGC-3' 5'-CCATCTCAGCTACTGGAAAACATTC-3' BHQ probe: RB1-2727 BHQ 5'-FAM-CAGCACTTCTTTTGAGCACACGGTCG-BHQ1 - 3' MGB probe: RB1-2727 MGB: 5'-FAM-CAGCACTTCTTTTGAGCAC-MGBNFQ-3' Primers for SOE: RB1 2750 SOE F RB1 2750 SOE R pRB1 F pRB1 R 5'-GTGCTGAACTAACCAACGCTCCGAAACGACTGAA -3' 5'-TTCAGTCGTTTCGGAGCGTTGGTTAGTTCAGCACTTC-3' 5'-AGGTTGCTAACTATGAAACACTGGC-3' 5'-CCATCTCAGCTACTGGAAAACATTC-3' Primers for RB1-IPC RB1 2750 IPC R RB1-2672 F 5'-TCGTTTCGGAGCGTTGGTTAG 3' 5'-CCAGAAAATAAATCAGATGGTATGTAACA-3' Mitochondrial Target: Spans Over the Genes for Transfer Ribonucleic Acid Lysine and ATP Synthase 8 Primers for cloning: pMt F 5'-GGGTATACTACGGTCAATGCTCTGA-3' pMt R 5'-CAATGAATGAAGCGAACAGATTTTC-3' Primers for mt target: (5) mt-8294 F mt-8436 R 5'-CCACTGTAAAGCTAACTTAGCATTAACC-3' 5'-GTGATGAGGAATAGTGTAAGGAGTATGG-3' BHQ-Probe: mt-8345 BHQ 5'-JOE-CCAACACCTCTTTACAGTGAAATGCCCCA-BHQ1 - 3' MGB-Probe: mt-8345 MGB 5'-VIC - CCA ACA CCT CTT TAC AGT GAA-MGBDQ-3' Primers for SOE: mt8294 SOE F mt8294 SOE R pMt F pMt R 5'- GCCCAGTGTAGAGCTATGTTAGCATTTAGGTTTTAAGTTAA-3' 5'- TAAAACCTAAATGCTAACATAGCTCTACACTGGGCTCTAGAG-3' 5'-GGGTATACTACGGTCAATGCTCTGA-3' 5'-CAATGAATGAAGCGAACAGATTTTC-3' Primers for mt-IPC mt8294 IPC F mt-8436 R 5'-CCAGTGTAGAGCTATGTTAGCATTTAGG-3' 5' - GTGATGAGGAATAGTGTAAGGAGTATGG-3' 22 Köchl, Niederstätter, and Parson 4.
1. For each set of samples to be quantified in one experiment, we routinely include a twofold serial dilution of the female DNA standard (from 1000 pg to 8 pg) to generate the standard AMG-X quantification curve for quantification of both male and female DNA. At least two male DNA controls (2500 pg and 250 pg) should also be included on each Quantification of AMG Gene Copies 35 experiment to monitor the performance of the AMGY-VIC detector or alternatively a standard AMG-Y quantification curve can be generated if a twofold serial dilution of a male DNA standard is included (see Note 3).