Prior research has shown that chromosomal positioning within the nucleus—whether, for example, a gene is adjacent to the nuclear membrane, or one chromosomal region is near another—can influence gene expression [1−5]. Such localization can also affect how and where chromosomal rearrangement occurs—chromosomal breakage followed by rejoining at incorrect positions can lead to disease .
Multicolor visualization of large genomic regions
To better understand the three dimensional organization of the genome, scientists in the laboratory of Dr Ting Wu (Genetics Department, Harvard School of Medicine, Boston, MA, USA) have developed a new tool called Oligopaints for fluorescent in situ hybridization (FISH) imaging of chromosomes . These short, fluorescently-labeled oligonucleotides enable single and multicolor visualization of genomic regions ranging in size from tens of kilobases to megabases, and can be used in fixed cultured cells, fixed tissue, and on metaphase chromosome spreads. A few design tricks (see below) make these probes particularly appealing—the probes are single-stranded and strand-specific, renewable via amplification, and include a non-homologous “tail” sequence to which a precise number of fluorescent dyes or other labels can be attached. They are also kept short to facilitate entry into the nucleus.
Designing Oligopaint probes
Libraries of hundreds to hundreds of thousands of oligos are designed using OligoArray software  that identifies appropriate genomic target sequences within each chromosome. The researchers use 74-base oligos that have just 32 bases of homology to each target and, importantly, they have mined the entirety of the human, mouse, Drosophila, nematode, and Arabidopsis genomes for suitable Oligopaint targets and made them available through their Oligopaints website; regardless of which genome, the software identifies an average of ~10 targets per kilobase. Pools of identified oligo sequences are synthesized on a solid support and released. Each of the resulting oligos contains the 32-base region of genomic target homology with flanking 21-base priming sequences. One of the amplification primers contains a 5’ fluorophore and the other contains a nicking site, positioned at the boundary with the 32-base region of homology. After amplification, an enzymatic nicking reaction removes the latter priming sequence, resulting in a 53- base, single-stranded, fluorophore-labeled oligo that can be isolated by denaturing gel electrophoresis.
Researchers can design Oligopaints to target specifically either strand of the chromosomal DNA, which will facilitate studies of transcription as well as other strand-specific aspects of chromosome biology. These new probes can also be used to simultaneously label a chromosomal region (Figure 1) as well as transcripts from that same region. Finally, hundreds of thousands of oligo probes can be generated in parallel, enabling megabases of the genome to be ‘painted’ at a fraction of current commercial costs.
Figure 1. Multicolor Mapping of Chromosomal Arm.
IDT supplied the amplification primers and, in some cases, the libraries of oligos, for development and testing of Oligopaints. Dr Wu recalls, “While we love the products IDT provides, what I really want to highlight is the scientific discussions and encouragement the IDT scientists gave us. It was helpful for me, and extremely motivating for the people in my lab. Their supportiveness pushed our thinking about the project and reminded us of why we went into science.”
IDT products that can facilitate your research
You can order up to 1 µmol desalted, custom synthesized DNA oligonucleotides and they will be shipped to you the next business day (larger scales are shipped within 5 business days). You can also specify whether to receive them dried down or hydrated, and whether you want them already annealed. Every IDT oligonucleotide you order is deprotected and desalted to remove small molecule impurities. Your oligos are quantified twice by UV spectrophotometry to provide an accurate measure of yield. Standard oligos are also assessed by mass spectrometry for quality you can count on.
Learn more or order now.
Custom dsDNA fragments
Rather than annealing oligonucleotides to obtain dsDNA fragments, when your fragment size is 125 bp or longer, it might make more sense to order gBlocks® Gene Fragments. gBlocks Gene Fragments are double-stranded, sequence-verified, DNA genomic blocks, 125–2000 bp in length, that can be shipped in 2–5 working days for affordable and easy gene construction or modification. These dsDNA fragments have been used in a wide range of applications including CRISPR-mediated genome editing, antibody research, codon optimization, mutagenesis, and aptamer expression. They can also be used for generating qPCR standards.
Learn more about gBlocks Gene Fragments at www.idtdna.com/gblocks.
- Ferrai C, de Castro IJ, et al. (2010) Gene positioning. Cold Spring Harb Perspect Biol, 2:a000588.
- Henikoff S. (2010) Summary: The nucleus—a close-knit community of dynamic structures. Cold Spring Harb Symp Quant Biol, 75:607−615.
- Meister P, Mango SE, Gasser SM. (2011) Locking the genome: nuclear organization and cell fate. Curr Opin Genet Dev, 21:167−174.
- Rajapakse I and Groudine M. (2011) On emerging nuclear order. J Cell Biol, 192:711−721.
- Holwerda S and de Laat W. (2012) Chromatin loops, gene positioning, and gene expression. Front Genet, 3:217.
- Wijchers PJ and de Laat W. (2011) Genome organization influences partner selection for chromosomal rearrangements. Trends Genet, 27:63−71.
- Beliveau BJ, Joyce EF, et al. (2012) Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes. Proc Natl Acad Sci USA, 109(52):21301–21306.
- Rouillard JM, Zuker M, and Gulari E. (2003) OligoArray 2.0: Design of oligonucleotide probes for DNA microarrays using a thermodynamic approach. Nucleic Acids Res, 31(12):3057−3062.
Author: Ellen Prediger, PhD, is Director of Scientific Communication at IDT.
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