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In Silico Biology 5, 0043 (2005); ©2005, Bioinformation Systems e.V.  



Accessing optimal primer distance from insert

Francisco Prosdocimi1 and J. Miguel Ortega2,*

1 Departamento de Biologia Geral, ICB-UFMG, 31270-010, Belo Horizonte/MG, Brazil
   Email: franc@icb.ufmg.br
2 Departamento de Bioquímica e Imunologia, ICB-UFMG, 31270-010, Belo Horizonte/MG, Brazil
   Email: miguel@icb.ufmg.br

* Corresponding author


Edited by H. Michael; received June 24, 2005; revised and accepted September 05, 2005; published September 24, 2005


Abstract

When building either DNA or cDNA libraries, a researcher looks at the vector multiple cloning site and chooses which restriction enzyme(s) will be used to clone the inserts. Although this procedure does not seem to be important, the accurate choosing of primer to insert distance can save time and money from genome and transcriptome projects. Here, 846 single-pool pUC18 sequences were produced and compared with the pUC18 consensus using local alignment tools. Data show that reads often contain 0-20 miscalled bases at the beginning of read and noise to signal transition is frequently found at 46-54 bases from the first 3 base downstream the sequencing primer. For SWAT-based approaches, 60 bases was the distance where over 90% of the sequences provided reliable information, presenting 13 vector bases on average. Looking at the data, it is possible to choose the most appropriate primer to insert distance for many applications.


Keywords: pUC18, cloning vector, Smith-Waterman, BLAST, primer positioning, insert, MegaBACE sequencer