Dataset Information

PCR amplification of tandemly repeated DNA: analysis of intra- and interchromosomal sequence variation and homologous unequal crossing-over in human alpha satellite DNA.

ABSTRACT: Tandemly repeated DNA can comprise several percent of total genomic DNA in complex organisms and, in some instances, may play a role in chromosome structure or function. Alpha satellite DNA is the major family of tandemly repeated DNA found at the centromeres of all human and primate chromosomes. Each centromere is characterized by a large contiguous array of up to several thousand kb which can contain several thousand highly homogeneous repeat units. By using a novel application of the polymerase chain reaction (repPCR), we are able to amplify a representative sampling of multiple repetitive units simultaneously, allowing rapid analysis of chromosomal subsets. Direct sequence analysis of repPCR amplified alpha satellite from chromosomes 17 and X reveals positions of sequence heterogeneity as two bands at a single nucleotide position on a sequencing ladder. The use of TdT in the sequencing reactions greatly reduces the background associated with polymerase pauses and stops, allowing visualization of heterogeneous bases found in as little as 10% of the repeat units. Confirmation of these heterogeneous positions was obtained by comparison to the sequence of multiple individual cloned copies obtained both by PCR and non-PCR based methods. PCR amplification of alpha satellite can also reveal multiple repeat units which differ in size. Analysis of repPCR products from chromosome 17 and X allows rapid determination of the molecular basis of these repeat unit length variants, which appear to be a result of unequal crossing-over. The application of repPCR to the study of tandemly repeated DNA should allow in-depth analysis of intra- and interchromosomal variation and unequal crossing-over, thus providing insight into the biology and genetics of these large families of DNA.

SUBMITTER: Warburton PE

PROVIDER: S-EPMC334470 | biostudies-other | 1992 Nov

REPOSITORIES: biostudies-other

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PCR amplification of tandemly repeated DNA: analysis of intra- and interchromosomal sequence variation and homologous unequal crossing-over in human alpha satellite DNA.

Warburton P E PE Willard H F HF

Nucleic acids research 19921101 22

Tandemly repeated DNA can comprise several percent of total genomic DNA in complex organisms and, in some instances, may play a role in chromosome structure or function. Alpha satellite DNA is the major family of tandemly repeated DNA found at the centromeres of all human and primate chromosomes. Each centromere is characterized by a large contiguous array of up to several thousand kb which can contain several thousand highly homogeneous repeat units. By using a novel application of the polymera ...[more]

PMID: 1461735

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Project description:BackgroundFunctional and morphological studies of tandem DNA repeats, that combine high portion of most genomes, are mostly limited due to the incomplete characterization of these genome elements. We report here a genome wide analysis of the large tandem repeats (TR) found in the mouse genome assemblies.ResultsUsing a bioinformatics approach, we identified large TR with array size more than 3 kb in two mouse whole genome shotgun (WGS) assemblies. Large TR were classified based on sequence similarity, chromosome position, monomer length, array variability, and GC content; we identified four superfamilies, eight families, and 62 subfamilies - including 60 not previously described. 1) The superfamily of centromeric minor satellite is only found in the unassembled part of the reference genome. 2) The pericentromeric major satellite is the most abundant superfamily and reveals high order repeat structure. 3) Transposable elements related superfamily contains two families. 4) The superfamily of heterogeneous tandem repeats includes four families. One family is found only in the WGS, while two families represent tandem repeats with either single or multi locus location. Despite multi locus location, TRPC-21A-MM is placed into a separated family due to its abundance, strictly pericentromeric location, and resemblance to big human satellites. To confirm our data, we next performed in situ hybridization with three repeats from distinct families. TRPC-21A-MM probe hybridized to chromosomes 3 and 17, multi locus TR-22A-MM probe hybridized to ten chromosomes, and single locus TR-54B-MM probe hybridized with the long loops that emerge from chromosome ends. In addition to in silico predicted several extra-chromosomes were positive for TR by in situ analysis, potentially indicating inaccurate genome assembly of the heterochromatic genome regions.ConclusionsChromosome-specific TR had been predicted for mouse but no reliable cytogenetic probes were available before. We report new analysis that identified in silico and confirmed in situ 3/17 chromosome-specific probe TRPC-21-MM. Thus, the new classification had proven to be useful tool for continuation of genome study, while annotated TR can be the valuable source of cytogenetic probes for chromosome recognition.

Project description:Polyploidization has played an important role in the evolution of vertebrates, particularly at the base of Teleostei-an enormously successful ray-finned fish group with additional genome doublings on lower taxonomic levels. The investigation of post-polyploid genome dynamics might provide important clues about the evolution and ecology of respective species and can help to decipher the role of polyploidy per se on speciation. Few studies have attempted to investigate the dynamics of repetitive DNA sequences in the post-polyploid genome using molecular cytogenetic tools in fishes, though recent efforts demonstrated their usefulness. The demonstrably monophyletic freshwater loach family Botiidae, branching to evolutionary diploid and tetraploid lineages separated >25 Mya, offers a suited model group for comparing the long-term repetitive DNA evolution. For this, we integrated phylogenetic analyses with cytogenetical survey involving Giemsa- and Chromomycin A3 (CMA3)/DAPI stainings and fluorescence in situ hybridization with 5S/45S rDNA, U2 snDNA and telomeric probes in representative sample of 12 botiid species. The karyotypes of all diploids were composed of 2n = 50 chromosomes, while majority of tetraploids had 2n = 4x = 100, with only subtle interspecific karyotype differences. The exceptional karyotype of Botia dario (2n = 4x = 96) suggested centric fusions behind the 2n reduction. Variable patterns of FISH signals revealed cases of intraspecific polymorphisms, rDNA amplification, variable degree of correspondence with CMA3+ sites and almost no phylogenetic signal. In tetraploids, either additivity or loci gain/loss was recorded. Despite absence of classical interstitial telomeric sites, large blocks of interspersed rDNA/telomeric regions were found in diploids only. We uncovered different molecular drives of studied repetitive DNA classes within botiid genomes as well as the advanced stage of the re-diploidization process in tetraploids. Our results may contribute to link genomic approach with molecular cytogenetic analyses in addressing the origin and mechanism of this polyploidization event.

Project description:Four unrelated families with the same unbalanced translocation der(4)t(4;11)(p16.2;p15.4) were identified. Both of the breakpoint regions in 4p16.2 and 11p15.4 were narrowed to large ~359-kb and ~215-kb low-copy repeat (LCR) clusters, respectively, by aCGH and SNP array analyses. DNA sequencing enabled mapping the breakpoints of one translocation to 24-bp within interchromosomal paralogous LCRs of ~130-kb in length and 94.7% DNA sequence identity located in olfactory receptor gene clusters, indicating nonallelic homologous recombination (NAHR) as the mechanism for translocation. To investigate the potential involvement of interchromosomal LCRs in recurrent chromosomal translocation formation, we performed computational genome-wide analyses and identified 5292 interchromosomal LCR substrate pairs, > 5-kb in size and sharing > 94% sequence identity that can potentially mediate chromosomal translocations. Additional proof for interchromosomal NAHR mediated translocation formation was provided by sequencing the breakpoints of another recurrent translocation, der(8)t(8;12)(p23.1;p13.31). The NAHR sites were mapped within 55-bp in ~7.8-kb paralogous subunits of 95.3% sequence identity located in the ~579-kb (chr8) and ~287-kb (chr12) LCR clusters. We demonstrate that NAHR mediates recurrent constitutional translocations throughout the human genome and provide a computationally determined genome-wide “recurrent translocation map”. Affymetrix Genome-Wide SNP Array 6.0 arrays (Affymetrix, Santa Clara, California) were employed to define the breakpoints on chromosomes 4, 8, 11, and 12. Analysis was performed according to the Genome-Wide Human SNP Nsp/Sty Assay Kit 5.0/6.0 protocol provided by the supplier. The arrays were scanned using a GeneChip® Scanner 3000 7G (Affymetrix, Inc.) and results were analyzed using Genotyping Console version 2.1 software. patient 1: Version 5 BAC array contained 853 BAC/PAC clones designed to cover genomic regions of 75 known genomic disorders, all 41 subtelomeric regions, and 43 pericentromeric regions. For each patient sample, two experiments were performed with reversal of the dye labels for the control and test samples. patient 2: Version 5.1 BAC array contained 853 BAC/PAC clones designed to cover genomic regions of 75 known genomic disorders, all 41 subtelomeric regions, and 43 pericentromeric regions. For each patient sample, two experiments were performed with reversal of the dye labels for the control and test samples. patient 3: The BAC emulated Version 6.1 OLIGO array was comprised of approximately 42,460 oligonucleotides representing 1400 BAC clones, covering ~150 genomic disorders, all 41 subtelomeric regions up to 12 Mb and 43 pericentromeric regions with backbone coverage of every chromosome at the 650-band level of cytogenetic resolution (http://www.bcm.edu/geneticlabs/cma/tables.html). The 42.46K oligonucleotides (oligos) were selected from initial testing of 105,000 oligos derived from the Agilent eArray library with strict selection criteria and removal of repetitive sequences to ensure optimal performance with greater dynamic range. This targeted 42.46 K OLIGO array (V6 OLIGO) corresponds to genomic regions covered by the V6 BAC arrays and was manufactured in a 4 x 44K format with an average of 28-30 oligos per region previously covered by a single BAC clone. patient 5: The BAC emulated Version 6.3 OLIGO array was comprised of approximately 43,580 oligonucleotides representing 1400 BAC clones, covering ~150 genomic disorders, all 41 subtelomeric regions up to 12 Mb and 43 pericentromeric regions with backbone coverage of every chromosome at the 650-band level of cytogenetic resolution (http://www.bcm.edu/geneticlabs/cma/tables.html). This targeted OLIGO array corresponds to genomic regions covered by the V6 BAC arrays and was manufactured in a 4 x 44K format with an average of 28-30 oligos per region previously covered by a single BAC clone. patient 6: The BAC emulated Version 6.4 OLIGO array was comprised of approximately 43,700 oligonucleotides representing 1400 BAC clones, covering ~150 genomic disorders, all 41 subtelomeric regions up to 12 Mb and 43 pericentromeric regions with backbone coverage of every chromosome at the 650-band level of cytogenetic resolution (http://www.bcm.edu/geneticlabs/cma/tables.html). This targeted OLIGO array corresponds to genomic regions covered by the V6 BAC arrays and was manufactured in a 4 x 44K format with an average of 28-30 oligos per region previously covered by a single BAC clone. Patient 4 was a known t(4;11) translocation patient form previous report and only ran on Affymetrix array.

Dataset Information

PCR amplification of tandemly repeated DNA: analysis of intra- and interchromosomal sequence variation and homologous unequal crossing-over in human alpha satellite DNA.

Publications

PCR amplification of tandemly repeated DNA: analysis of intra- and interchromosomal sequence variation and homologous unequal crossing-over in human alpha satellite DNA.

Similar Datasets

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