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Imaging as a tool for improving length and accuracy of sequence analysis in automated fluorescence‐based DNA sequencing
Author(s) -
Sanders Jane Z.,
Petterson A. Albert,
Hughes Peter J.,
Connell Charles R.,
Raff Malcolm,
Menchen Steven,
Hood Leroy E.,
Teplow David B.
Publication year - 1991
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150120103
Subject(s) - dna , dna sequencing , resolution (logic) , signal (programming language) , limiting , sequence analysis , sequence (biology) , artificial intelligence , computational biology , computer science , pattern recognition (psychology) , biological system , biology , genetics , mechanical engineering , engineering , programming language
A new method of signal analysis for automated fluorescence‐based DNA sequencing is presented. Signal resolution is a limiting factor in obtaining accurate sequence information beyond 400–450 nucleotides per gel lane. We have developed a computer program for the imaging of DNA bands in sequencing gels. The image analysis shows that distortions in the shapes of the bands decrease resolution of peaks observed in the standard data plots. Reconstruction of the undistorted band shape prior to signal analysis substantially improves the resolution of peaks and may improve the accuracy and length of the contiguous sequence read. Image analysis identified other factors limiting the accuracy and length of automated DNA sequence analysis and provided a tool for evaluating various remedies. Our techniques should also be applicable in other systems, for example, in gel electrophoresis of proteins and DNA restriction fragments, and in scranning densitometry.