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Description COMT NlaIII Polymorphism Procedure NlaIII CURRENT PROTOCOL Template: Genomic DNA 300 ng/reaction volume of 25 µl Primers: Forward Primer: 5'-GCCCGCCTGCTGTCACC-3', Final Concentration = 200 nM Reverse Primer: 5'-CTGAGGGGCCTGGTGATAGTG-3', Final Concentration = 200 nM dNTPs: Final Concentration = 200 µM each Perkin Elmer Buffer II: Final Concentration = 1X MgCl2: Final Concentration = 1.5 mM Taq DNA Polymerase: 1.0 Units/reaction (Perkin Elmer AmpliTaq DNA polymerase) PCR Cycling Profile: 94oC , 1' [94oC , 30" - 60oC , 30" - 72oC , 30"] x 30 cycles 72oC , 10' NlaIII digestion: Nine µl of each PCR reaction was digested with 3 units of NlaIII restriction enzyme (Boehringer-Mannheim or New England Biolabs) in a total of 11 µl. Only 0.2 µl of the 10X restriction buffer recommended by the supplier was added to each digestion reaction. Product Size: NlaIII 238 bp -----------> 114 bp COMT*H, uncut allele, or 96 bp and 18 bp COMT*L allele, cut allele. Constant bands: 70 bp and 54 bp II. NlaIII (modified from Lachman et al, 1996) OLD PROTOCOL The first six 5' bases of COMT3R primer (from Lachman et al., 1996) do not match the genomic sequence now available from the Human Genome Sequencing (HGS) project. The primers used in the current protocol (see above) fully agree with genomic sequence from the HGS project. Template: Genomic DNA 100-200 ng/reaction volume of 25 ml Primer 1. COMT2F 5'--6-FAM-CTCATCACCATCGAGATCAACC--3' 250 nM/reaction Primer 2. COMT3R 5'--GATGACCCTGGTGATAGTGG--3' 250 nM/reaction dNTPs: Final Concentration 250 µM each Buffer: Final Concentration 2.25 mM MgCl2, 50 mM KCL, 10 mM Tris.HCl pH 8.3, 0.01% gelatin Taq DNA Polymerase: 1.0 Units/reaction (Perkin Elmer AmpliTaq DNA polymerase) PCR Cycling Profile: 80oC , 5' 94oC , 2' [94oC , 1' - 57oC , 30" - 72oC , 30"] ×30 cycles 72oC , 3' Special Instructions: Twenty ml of PCR reaction cocktail (Primers, dNTPs, and Buffer) was added to 1 µl genomic DNA at 100 ng/µl. The reactions were heated at 80oC for 4.5', then paused at 80oC while 4 ml of 0.25 U/ml of Taq polymerase enzyme was added. The temperature was kept at 80oC for 30" longer, then the cycle continued as above starting at the 94oC , 2' step. NlaIII digestion: Nine ml of each PCR reaction was digested with 3 units of NlaIII restriction enzyme (Boehringer-Mannheim or New England Biolabs) in a total of 11 ml. Only 0.2 ml of the 10X restriction buffer recommended by the supplier was added to each digestion reaction. Product Size: NlaIII 228 bp ----------->85 bp COMT*H, uncut allele, or 69 bp COMT*L allele fluoresce Constant bands: 71 bp, 54 bp, and 18 bp do not fluoresce Lachman HM, Morrow B, Shprintzen R, Veit S, Parsia SS, Faedda G, et al (1996): Association of codon 108/158 catechol-O-methyltransferase gene polymorphism with the psychiatric manifestations of velo-cardio-facial syndrome. Am. J. Med. Genet. 67:468-472. Palmatier, M.A., A. Min Kang, and K.K. Kidd, 1999. Global variation in the frequencies of functionally different catechol-O-methyltransferase alleles. Biological Psychiatry 46:557-567. Recipes Supplies Tips (责任编辑：泉水) |
COMT NlaIII Polymorphism
时间:2005-07-18 00:00来源:Scienceboard.net 作者:admin 点击:
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