Home LiteratureArticle Details
PMID: 11997346 Published · ppublish English Journal Article Research Support, U.S. Gov't, Non-P.H.S.

Integration of Cot analysis, DNA cloning, and high-throughput sequencing facilitates genome characterization and gene discovery.

Genome research ·Vol. 12 ·No. 5 ·2002-05-00 ·Pages 795-807

Peterson DG, Schulze SR, Sciara EB, Lee SA, Bowers JE, Nagel A, Jiang N, Tibbitts DC, Wessler SR, Paterson AH

Abstract

Cot-based sequence discovery represents a powerful means by which both low-copy and repetitive sequences can be selectively and efficiently fractionated, cloned, and characterized. Based upon the results of a Cot analysis, hydroxyapatite chromatography was used to fractionate sorghum (Sorghum bicolor) genomic DNA into highly repetitive (HR), moderately repetitive (MR), and single/low-copy (SL) sequence components that were consequently cloned to produce HRCot, MRCot, and SLCot genomic libraries. Filter hybridization (blotting) and sequence analysis both show that the HRCot library is enriched in sequences traditionally found in high-copy number (e.g., retroelements, rDNA, centromeric repeats), the SLCot library is enriched in low-copy sequences (e.g., genes and "nonrepetitive ESTs"), and the MRCot library contains sequences of moderate redundancy. The Cot analysis suggests that the sorghum genome is approximately 700 Mb (in agreement with previous estimates) and that HR, MR, and SL components comprise 15%, 41%, and 24% of sorghum DNA, respectively. Unlike previously described techniques to sequence the low-copy components of genomes, sequencing of Cot components is independent of expression and methylation patterns that vary widely among DNA elements, developmental stages, and taxa. High-throughput sequencing of Cot clones may be a means of "capturing" the sequence complexity of eukaryotic genomes at unprecedented efficiency.

MeSH Terms
Base Composition/genetics Blotting, Southern/methods Chromosomes, Artificial, Bacterial/genetics Cloning, Molecular/methods Expressed Sequence Tags GC Rich Sequence/genetics Gene Dosage Genes, Plant Genetic Markers/genetics Genome, Plant Genomic Library Molecular Sequence Data Nucleic Acid Denaturation Nucleic Acid Hybridization Plant Proteins/genetics Poaceae/genetics Repetitive Sequences, Nucleic Acid/genetics Sequence Analysis, DNA/methods Temperature
Chemicals
Genetic Markers Plant Proteins kafirin protein, Sorghum bicolor
Authors & Affiliations
10 authors, click to expand affiliations / ORCID
Peterson Daniel G
Center for Applied Genetic Technologies and Department of Crop and Soil Sciences, University of Georgia, Athens, Georgia 30602, USA. dgp@arches.uga.edu
Schulze Stefan R
Sciara Erica B
Lee Scott A
Bowers John E
Nagel Alexander
Jiang Ning
Tibbitts Deanne C
Wessler Susan R
Paterson Andrew H
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Article Info
Journal
Genome research
Abbr.
Genome Res
ISSN
1088-9051
DOI
10.1101/gr.226102
Email
dgp@arches.uga.edu
Published
2002-05-00
Pages
795-807
Language
English
Region
United States
NLM ID
9518021
PMCID
PMC186575
Subset
IM
Databases
GENBANK
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PubMed source DOI: 10.1101/gr.226102 PMC Full Text View journal details
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