Acknowledgements
The article is dedicated to Ilia V. Soloviev. I would like to express my gratitude to Prof. Svetlana G Vorsanova and Prof. Yuri B Yurov for helping in the preparation of this chapter. This work was supported by the Russian Science Foundation (Grant #14-35-00060).
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Chapter 14
High Resolution Fiber-Fluorescence In Situ Hybridization
Christine J. Ye and Henry H. Heng
Abstract
High resolution fi ber-Fluorescence in situ hybridization (FISH) is an advanced FISH technology that can effectively bridge the resolution gap between probe hybridizing on DNA molecules and chromosomal regions. Since various types of DNA and chromatin fi bers can be generated refl ecting different degrees of DNA/chromatin packaging status, fi ber-FISH technology has been successfully used in diverse molecular cytogenetic/cytogenomic studies. Following a brief review of this technology, including its major develop- ment and increasing applications, typical protocols to generate DNA/chromatin fi ber will be described, coupled with rationales, as well as technical tips. These released DNA/chromatin fi bers are suitable for an array of cytogenetic/cytogenomic analyses.
Key words DNA fi ber-FISH , Chromatin fi ber-FISH , Halo FISH , Stretched chromatin fi ber-FISH , Free chromatin-FISH
1 Introduction
Despite the fact that free chromatin and defective mitotic fi gures (DMFs) were initially identifi ed over 30 years ago for the purpose of analyzing high order chromosomal structures, it was the desire and requirement of high resolution FISH technology for physical mapping that introduced and promoted the development of fi ber- FISH methodology [ 1 – 5 ]. The rationale of applying FISH detec- tion on released chromatin fi bers is straightforward: (1) Experimentally released chromatin fi bers are less condensed than native chromosomes and even interphase chromatin, and different degrees of decondensation can be achieved based on the releasing conditions; (2) The distance between hybridized targets can be measured on stretched linear DNA/chromatin fi ber so that quan- titative measurements can be acquired.
The free chromatin FISH was initially introduced to improve the resolution of interphase FISH and meiotic chromatin FISH [ 6 , 7 ]. The idea was quickly appreciated by different groups and vari- ous protocols were developed within a short period [ 8 – 14 ].
Modifi ed protocols were established carrying different names,
including free DNA FISH , DNA halo FISH , extended chromatin/
DNA FISH , direct visual hybridization ( DIRVSH ), molecular combing or DNA/chromosome combing, and quantitative DNA fi ber-FISH. Despite these different names, the major difference among these protocols is the means of releasing/preparing chro- matin or DNA fi bers on microscopic slides prior to probe hybrid- ization. A variety of high-resolution FISH methods have been collectively referred to as high-resolution fi ber FISH [ 15 , 16 ]. The key consideration of selecting a protocol depends on the mapping resolution, target coverage, involvement of chromatin structure such as DNA/protein codetection, data interpretation, and avail- able equipment/materials/reagents. For example, DNA fi bers are better for the highest resolution mapping within a small region of the genome. In contrast, the term “chromatin fi ber” describes chromatin released from the nucleus without striping most chro- matin proteins; these fi bers generally correspond to 30-nm struc- tures. With a certain degree of preserved high order structure, released chromatin fi bers offer an advantage in chromatin struc- tural studies covering a relatively larger mapping region.
The introduction of fi ber-FISH methodologies has greatly advanced the analyses of human, animal, and plant genomes, as well as lower eukaryote genomes, such as fungi and protozoan parasites [ 17 – 21 ]. Examples include: the sequencing gap estima- tion for the Human Genome Project; the study of copy number polymorphism among different individuals [ 22 ]; the determina- tion of order and orientation of groups of genes/ESTs or DNA fragments [ 23 ]; the quantifi cation of the sizes of duplicated or amplifi ed fragments of special genes, chromosomal regions, or integrated foreign inserts [ 24 , 25 ]; the identifi cation or exclusion of genes or chromosomal regions defi ned by particular genetic markers [ 26 ]; the comparison of evolutionary conserved genomic regions among various species or cell lines [ 27 , 28 ]; the illustra- tion of multiple repetitive sequences within particular genomic regions and the direct visualization of genome organization [ 29 ];
the length measurement of telomeres or centromeres for the study of chromosomal packaging both in mitotic and meiotic cells [ 30 – 32 ]; and the study of DNA replication status and repair processing combined with codetection of DNA repair proteins [ 33 ]. Equally important, fi ber-FISH has successfully applied to analyze genetic aberrations of human diseases, such as mapping translocation breakpoints [ 34 , 35 ]; determining the number and orientation of duplicated genes that are responsible for Pelizaeus- Merzbacher disease [ 36 ]; developing probe sets covering many critical chromosomal regions involving diseases, which are essen- tial for disease diagnosis when translocation or deletion/ duplication occurs [ 37 , 38 ] and illustrating the copy number variation among individual [ 22 ].
Realizing it is the entire set of chromosomes not just the indi- vidual genes defi ning the blueprint and function of cells, the karyo- type represents an independent type of genetic coding, which preserves the topological relationship for gene interaction for a given species [ 39 – 41 ]. Therefore, the cancer cytogenetic analysis becomes increasingly important as genome heterogeneity is the key driver of cancer evolution [ 42 , 43 ]. Such knowledge calls for the departure from gene-centric concepts to a novel genome the- ory where the molecular cytogenetic and cytogenomic analyses are essential [ 44 , 45 ]. In particular, the systematic characterization of various types of chaotic genomes becomes a priority, and fi ber- FISH will play an increasingly important role.
In this chapter, a number of protocols are provided for each type of fi ber preparation to serve the purpose of diverse applica- tions. Even though different reagents are used in variable proto- cols, they all share the goal of releasing high quality chromatin/
DNA fi ber using less complicated procedures. By comparing dif- ferent protocols, readers can easily identify key steps and even modify them for their own experiments.
2 Materials