Polymerase chain reaction-based suppression of repetitive sequences in whole chromosome painting probes for FISH

Dugan, Lawrence; Pattee, Melissa S.; Williams, J F; Mike, y; Sorensen, Karen; Bedford, Joel S.; Christian, Allen T.

doi:10.1007/s10577-005-2349-7

Cited by 10 publications

(6 citation statements)

References 10 publications

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“…This region contains a variety of short interspersed elements (SINEs), causing nonspecific probe binding which is difficult to block. It was evident that blocking nonspecific‐target DNA sites on the genome was required and was accomplished with Cot‐1 DNA .…”

Section: Discussionmentioning

confidence: 99%

Development of locus specific sub‐clone separation by fluorescence in situ hybridization in suspension in chronic lymphocytic leukemia

Bailey

Macardle

et al. 2017

Cytometry Pt A

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Intra-tumor genetic heterogeneity is a hallmark of cancer. The ability to monitor and analyze these sub-clonal cell populations can be considered key to successful treatment, particularly in the modern era of targeted therapies. Although advances in sequencing technologies have significantly improved our ability to analyze the mutational landscape of tumors, this utility is reduced when considering small, but clinically significant sub-clones, that is, those representing <10% of the tumor burden. We have developed a high-throughput method that utilizes a 17-probe labeled bacterial artificial chromosome contig to quantify sub-clonal populations of cells based on deletion of a single locus. Chronic lymphocytic leukemia (CLL) cells harboring deletion of the short arm of chromosome 17 (del17p), an important prognostic marker for CLL were used to demonstrate the technique. Sub-clones of del17p cells were quantified and isolated from heterogeneous CLL populations using fluorescence in situ hybridization in suspension (FISH-IS) and the locus specific probe set. Using the combination of FISH-IS with the locus-specific probe set enables automated analysis of tens of thousands of cells, accurately quantifying and isolating cells carrying a del17p. Based on the fluorescence intensity of 17p probes, 17p (TP53) deleted cells were identified and sorted using flow cytometric techniques, and enrichment was demonstrated using single nucleotide polymorphism analysis. The ability to separate sub-clones of cells based on genetic heterogeneity, independent of the clone size, highlights the potential application of this method not only in the diagnostic and prognostic setting, but also as an unbiased approach to enable further detailed genetic analysis of the sub-clone with deep sequencing approaches. © 2017 International Society for Advancement of Cytometry.

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Section: Discussionmentioning

confidence: 99%

Development of locus specific sub‐clone separation by fluorescence in situ hybridization in suspension in chronic lymphocytic leukemia

Bailey

Macardle

et al. 2017

Cytometry Pt A

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“…An alternative technique would be to improve chromosome painting by generating the microdissection DNA probes with suppression of the PCR amplification of repetitive DNA sequences (Dugan et al 2005;Lucas et al 2006). This might allow the physical mapping of minority DNA sequences that remained obscured by repetitive DNA sequences after conventional amplification methods.…”

Section: Discussionmentioning

confidence: 99%

Microdissection and chromosome painting of X and B chromosomes in Locusta migratoria

et al. 2008

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Acquisition of knowledge of the nature and DNA content of B chromosomes has been triggered by a collection of molecular techniques, one of which, microdissection, has provided interesting results in a number of B chromosome systems. Here we provide the first data on the molecular composition of B chromosomes in Locusta migratoria, after microdissection of the B and X chromosomes, DNA amplification by one (B) or two (X) different methods, and chromosome painting. The results showed that B chromosomes share at least two types of repetitive DNA sequences with the A chromosomes, suggesting that Bs in this species most likely arose intraspecifically. One of these repetitive DNAs is located on the heterochromatic distal half of the B chromosome and in the pericentromeric regions of about half of the A chromosomes, including the X. The other type of repetitive DNA is located interspersedly over the non-centromeric euchromatic regions of all A chromosomes and in an interstitial part of the proximal euchromatic half of the B chromosome. Chromosome painting, however, did not provide results sufficiently reliable to determine, in this species, which A chromosome gave rise to the B; this might be done by detailed analysis of the microdissected DNA sequences.

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“…Plasmid DNA was purified from pooled colonies and inserts were amplified by PCR (M13 forward and reverse primer). A 100 μl reaction volume was prepared using 10 ng plasmid DNA, 10 μl 10× Ex Taq Buffer (contains 20 mM MgCl 2 ), 2.4 μl 25 mM dNTP solution, 0.6 μl of 100 μM M13 forward and reverse primer sets, 5 U TaKaRa Ex Taq DNA Polymerase and distilled, deionized H 2 O. Repeat-rich DNA (100 ng; human Cot-1 DNA) was also included in the reaction mixture to eliminate repetitive sequences by interfering with extension of the probe across repetitive sequences [19]. The temperature-time cycling profile was as follows: 95°C for 5 minutes followed by 20 cycles of 94°C for 1 minute, 55°C for 20 s and 72°C for 30 s. This was followed by 5 minutes at 72°C and a hold at 4°C until tubes were removed.…”

Section: Methodsmentioning

confidence: 99%

Detection of DNA fusion junctions for BCR-ABL translocations by Anchored ChromPET

2010

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Anchored ChromPET, a technique to capture and interrogate targeted sequences in the genome, has been developed to identify chromosomal aberrations and define breakpoints. Using this method, we could define the BCR-ABL1 translocation DNA breakpoint to a base-pair resolution in Philadelphia chromosome-positive samples. This DNA-based method is highly sensitive and can detect the fusion junction using samples from which it is hard to obtain RNA or cells where the RNA expression has been silenced.

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Polymerase chain reaction-based suppression of repetitive sequences in whole chromosome painting probes for FISH

Cited by 10 publications

References 10 publications

Development of locus specific sub‐clone separation by fluorescence in situ hybridization in suspension in chronic lymphocytic leukemia

Development of locus specific sub‐clone separation by fluorescence in situ hybridization in suspension in chronic lymphocytic leukemia

Microdissection and chromosome painting of X and B chromosomes in Locusta migratoria

Detection of DNA fusion junctions for BCR-ABL translocations by Anchored ChromPET

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