Abstract
BackgroundOver 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.ResultsIn 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2437-4) contains supplementary material, which is available to authorized users.
Highlights
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
We developed an approach for lampbrush chromosome microdissection, DNA or Complementary DNA (cDNA) amplification from the isolated material followed by highly specific fluorescent in situ hybridization (FISH)-probes generation and highthroughput sequencing
Microdissection of individual chromomeres and lateral loops from chicken lampbrush chromosomes Here we developed a workflow for lampbrush chromosome microdissection followed by locus-specific FISHprobe generation and high-throughput sequencing (Fig. 1)
Summary
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. Glass needle-based microdissection helps to obtain broad panels of locus-specific and whole-chromosome painting probes marking different chromosomal regions of various length and organization complexity (including the regions enriched by highlyrepetitive DNA). Such probes are widely used to identify the chromosomes, to map precisely the evolutionary and clinical chromosomal breakpoints, as well as to determine the nature of marker and derivative chromosomes [2,3,4,5,6,7,8,9,10]. It became possible to analyze dissected chromosomal regions using next-generation sequencing (NGS) technologies [23, 24] which offer great prospects for more detailed research of specific loci
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