title: Microdissection of lampbrush chromosomes as an approach for generation of locus-specific FISH-probes and samples for high-throughput sequencing.
abstract: BACKGROUND
  Over the past two decades, chromosome microdissection has been widely used in diagnostics and research enabling analysis of chromosomes and their regions through probe generation and establishing of chromosome- and chromosome region-specific DNA libraries. However, relatively small physical size of mitotic chromosomes limited the use of the conventional chromosome microdissection for investigation of tiny chromosomal regions.
  
  
  RESULTS
  In the present study, we developed a workflow for mechanical microdissection of giant transcriptionally active lampbrush chromosomes followed by the preparation of whole-chromosome and locus-specific fluorescent in situ hybridization (FISH)-probes and high-throughput sequencing. In particular, chicken (Gallus g. domesticus) lampbrush chromosome regions as small as single chromomeres, individual lateral loops and marker structures were successfully microdissected. The dissected fragments were mapped with high resolution to target regions of the corresponding lampbrush chromosomes. For investigation of RNA-content of lampbrush chromosome structures, samples retrieved by microdissection were subjected to reverse transcription. Using high-throughput sequencing, the isolated regions were successfully assigned to chicken genome coordinates. As a result, we defined precisely the loci for marker structures formation on chicken lampbrush chromosomes 2 and 3. Additionally, our data suggest that large DAPI-positive chromomeres of chicken lampbrush chromosome arms are characterized by low gene density and high repeat content.
  
  
  CONCLUSIONS
  The developed technical approach allows to obtain DNA and RNA samples from particular lampbrush chromosome loci, to define precisely the genomic position, extent and sequence content of the dissected regions. The data obtained demonstrate that lampbrush chromosome microdissection provides a unique opportunity to correlate a particular transcriptional domain or a cytological structure with a known DNA sequence. This approach offers great prospects for detailed exploration of functionally significant chromosomal regions.
authors.affiliation: Department of Cytology and Histology, Saint Petersburg State University, Saint Petersburg, Russia. anna-zlotina@yandex.ru.
authors.forename: Anna
authors.initials: A
authors.lastname: Zlotina
authors.affiliation: Department of Cytology and Histology, Saint Petersburg State University, Saint Petersburg, Russia. t.v.kulikova@gmail.com.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Kulikova
authors.affiliation: Institute of Human Genetics, Jena University Hospital, Friedrich Schiller University, Jena, Germany. nadezda.kosyakova@med.uni-jena.de.
authors.forename: Nadezda
authors.initials: N
authors.lastname: Kosyakova
authors.affiliation: Institute of Human Genetics, Jena University Hospital, Friedrich Schiller University, Jena, Germany. Thomas.Liehr@med.uni-jena.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Liehr
authors.affiliation: Department of Cytology and Histology, Saint Petersburg State University, Saint Petersburg, Russia. alla.krasikova@gmail.com.
authors.forename: Alla
authors.initials: A
authors.lastname: Krasikova
mesh_terms: D000818:Animals; D002645:Chickens; D002874:Chromosome Mapping; D002875:Chromosomes / Q000648:ultrastructure*; D003582:Cytogenetics / Q000379:methods; D015342:DNA Probes; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing; D017404:In Situ Hybridization, Fluorescence; D042282:Microdissection; D017422:Sequence Analysis, DNA
chemical_list: D015342:DNA Probes