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Saturday, April 22, 2006 12:22:11 AM
Here you go buckshot, but I suggest you do a little more homework. Mykel may have made a couple of minor mistatements, but it's clear he has a good working knowledge. You my friend should stick to engineering:
http://investorshub.com/boards/read_msg.asp?message_id=10766897
PCR amplification (Editor's Note: You ARE familiar with the PCR process, correct?) was accomplished using pfu Turbo polymerase according to the manufacturer's guidelines (Stratagene, La Jolla, CA). We developed a program (T. FRUDAKIS, M. THOMAS, Z. GASKIN, K. VENKATESWARLU, K. SURESH CHANDRA, S. GINJUPALLI, S. GUNTURI, S. NATRAJAN, V. K. PONNUSWAMY and K. N. PONNUSWAMY, unpublished results) to design resequencing primers (Editor's Note: Are you familiar with primers, i.e. oligonucleotides, and their use?) in a manner respectful of homologous sequences in the genome, to ensure that we did not coamplify pseudogenes or amplify from within repeats. BLAST searches confirmed the specificity of all primers used. Amplification products were subcloned into the pTOPO (Invitrogen, San Diego) sequencing vector and 96 insert-positive colonies were grown for plasmid DNA isolation (the use of 670 individuals for the amplification step reduced the likelihood of an individual contributing more than once to this subset of 96 selected). We sequenced with an ABI3700 using PE Applied Biosystems BDT chemistry and we deposited the sequences into a commercial relational database system (iFINCH, Geospiza, Seattle). PHRED-qualified sequences were imported into the CLUSTAL X alignment program and the output of this was used with a second program that we developed (T. FRUDAKIS, M. THOMAS, Z. GASKIN, K. VENKATESWARLU, K. SURESH CHANDRA, S. GINJUPALLI, S. GUNTURI, S. NATRAJAN, V. K. PONNUSWAMY and K. N. PONNUSWAMY, unpublished results) to identify quality-validated discrepancies between sequences. We selected those for which at least two instances of PHRED identified variants that scored 24, and each of these SNPs discovered through resequencing were used for genotyping.
http://www2.carthage.edu/~pfaffle/hgp/ABI3700.html
Would you STILL like to claim that they've never sequenced anything? Boy, you are the same 'ole frog though...thick, yet obstinant.
Time to go now...have fun with your friends.
Later,
W2P
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