Nejvíce citovaný článek - PubMed ID 9668971
Subtraction with 3' modified oligonucleotides eliminates amplification artifacts in DNA libraries enriched for microsatellites
The application of flow cytometry and sorting (flow cytogenetics) to plant chromosomes did not begin until the mid-1980s, having been delayed by difficulties in preparation of suspensions of intact chromosomes and discrimination of individual chromosome types. These problems have been overcome during the last ten years. So far, chromosome analysis and sorting has been reported in 17 species, including major legume and cereal crops. While chromosome classification by flow cytometry (flow karyotyping) may be used for quantitative detection of structural and numerical chromosome changes, chromosomes purified by flow sorting were found to be invaluable in a broad range of applications. These included physical mapping using PCR, high-resolution cytogenetic mapping using FISH and PRINS, production of recombinant DNA libraries, targeted isolation of markers, and protein analysis. A great potential is foreseen for the use of sorted chromosomes for construction of chromosome and chromosome-arm-specific BAC libraries, targeted isolation of low-copy (genic) sequences, high-throughput physical mapping of ESTs and other DNA sequences by hybridization to DNA arrays, and global characterization of chromosomal proteins using approaches of proteomics. This paper provides a comprehensive review of the methodology and application of flow cytogenetics, and assesses its potential for plant genome analysis.
Procedures for flow cytometric analysis and sorting of mitotic chromosomes (flow cytogenetics) have been developed for chickpea (Cicer arietinum). Suspensions of intact chromosomes were prepared from root tips treated to achieve a high degree of metaphase synchrony. The optimal protocol consisted of a treatment of roots with 2 mmol/L hydroxyurea for 18 h, a 4.5-h recovery in hydroxyurea-free medium, 2 h incubation with 10 micromol/L oryzalin, and ice-water treatment overnight. This procedure resulted in an average metaphase index of 47%. Synchronized root tips were fixed in 2% formaldehyde for 20 min, and chromosome suspensions prepared by mechanical homogenization of fixed root tips. More than 4 x 10(5) morphologically intact chromosomes could be isolated from 15 root tips. Flow cytometric analysis of DAPI-stained chromosomes resulted in histograms of relative fluorescence intensity (flow karyotypes) containing eight peaks, representing individual chromosomes and/or groups of chromosomes with a similar relative DNA content. Five peaks could be assigned to individual chromosomes (A, B, C, G, H). The parity of sorted chromosome fractions was high, and chromosomes B and H could be sorted with 100% purity. PCR on flow-sorted chromosome fractions with primers for sequence-tagged microsatellite site (STMS) markers permitted assignment of the genetic linkage group LG8 to the smallest chickpea chromosome H. This study extends the number of legume species for which flow cytogenetics is available, and demonstrates the potential of flow cytogenetics for genome mapping in chickpea.
- MeSH
- buněčný cyklus MeSH
- chromozomy rostlin genetika MeSH
- Cicer genetika MeSH
- cytogenetika MeSH
- DNA rostlinná genetika metabolismus MeSH
- fyzikální mapování chromozomů metody MeSH
- genetická vazba MeSH
- genom rostlinný * MeSH
- hybridizace in situ fluorescenční MeSH
- indoly MeSH
- karyotypizace MeSH
- kořeny rostlin genetika MeSH
- metafáze MeSH
- mikrosatelitní repetice MeSH
- místa se sekvenční adresou MeSH
- mitóza MeSH
- polymerázová řetězová reakce MeSH
- průtoková cytometrie metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DAPI MeSH Prohlížeč
- DNA rostlinná MeSH
- indoly MeSH
