Key Points
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Whole-genome sequencing, positional cloning and comparative genomics mainly depend on the construction of high-quality physical maps.
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Current physical maps have been developed using agarose gel-based or, more recently, acrylamide gel-based techniques for fingerprinting large-insert clones.
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Fingerprinting methods based on fluorescent labelling are rich in information, have high throughput and produce more robust physical maps than traditional agarose gel-based methods.
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The use of high-throughput capillary electrophoresis machines and fluorescent fingerprinting methods makes physical map construction fast, efficient and largely automated.
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The proper use of statistics is required to produce high-quality physical maps, taking into account genome size and sequence complexity.
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There are methods to properly evaluate the quality and coverage of physical maps. The presence of undetected mis-assembled contigs can represent a serious problem if such methods are not applied.
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There are no non-model animal and plant species for which the same number and types of genomic resource are available as there are for humans, mice, rats, Arabidopsis thaliana and rice. This poses new challenges for the construction of whole-genome physical maps that require the adoption of refined clone-fingerprinting technologies.
Abstract
Physical maps provide an essential framework for ordering and joining sequence data, genetically mapped markers and large-insert clones in eukaryotic genome projects. A good physical map is also an important resource for cloning specific genes of interest, comparing genomes, and understanding the size and complexity of a genome. Although physical maps are usually taken at face value, a good deal of technology, molecular biology and statistics goes into their making. Understanding the science behind map building is important if users are to critically assess, use and build physical maps.
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Acknowledgements
We thank many colleagues at DuPont Crop Genetics–Genomics for helpful discussions on physical mapping. The physical mapping work in M.M.'s laboratory is supported by funding from Provincia Autonoma di Trento.
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Glossary
- BACTERIAL ARTIFICIAL CHROMOSOME
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(BAC). A cloning vector derived from a single-copy F-plasmid of Escherichia coli. Large genomic fragments (100–200 Kb) can be cloned into BACs, making them useful for constructing genomic libraries.
- YEAST ARTIFICIAL CHROMOSOME
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(YAC). A cloning vector system that can accommodate large genomic fragments (500–1,000 Kb). YACs are grown in yeast, and can be unstable and difficult to isolate in comparison to BACs.
- MINIMAL TILING PATH
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A minimal set of overlapping clones that together provides complete coverage across a genomic region.
- SULSTON CUTOFF SCORE
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A score that expresses the probability that the number of bands matched between any two clones being fingerprinted is a coincidence. Clones are considered to overlap if the score is below a user-supplied threshold (cutoff).
- RADIATION HYBRID CELL LINES
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A collection of cell lines, each of which is a clonal population of cells that are derived by the fusion of lethally X-irradiated donor cells with mammalian cells. Such cell lines can be used to create a physical map of the donor genome.
- METHYLATION FILTRATION
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A method that takes advantage of higher DNA methylation in repetitive than in low-single-copy sequences to selectively clone in Escherichia coli the latter (hypomethylated) ones that usually represent a gene-enriched fraction.
- HIGH COT SELECTION
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A method that takes advantage of faster re-naturation of repetitive than of low-single-copy sequences to select first and then clone in Escherichia coli the latter ones that represent a gene-enriched fraction.
- HAPPY MAPPING
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A simple method for ordering markers and determining the physical distances between them that uses subhaploid equivalents of randomly sheared DNA and requires the use of whole-genome amplification methods to perform multiple PCR reactions.
- SEQUENCE-TAGGED SITES
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(STS). Short (for example, <1,000 bp), unique sequence that is associated with a PCR assay that can be used to detect that site in the genome.
- INTERNAL SIZE STANDARD
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A set of DNA fragments of known size that are run in the same lane as the sample to be sized but distinguishable from the fragments of unknown size. Unlike the external size standards normally used on DNA gels, internal size standards allow for greater accuracy in sizing because they are not affected by lane-to-lane variation in the migration rate.
- STAR ACTIVITY
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The activity of restriction endonucleases under non-standard conditions that results in cleavage at sequences that are similar but not identical to their defined recognition sequence. The degree and type of this altered specificity varies among enzymes and reaction conditions.
- BONFERRONI CORRECTION
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A multiple-comparison correction to the significance level α that is used to avoid many spurious positives (type I errors) when several independent statistical tests are being performed simultaneously.
- LONG TERMINAL REPEAT RETROTRANSPOSONS
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(LTR retrotransposons). Transposable elements that move through an RNA intermediate, are related to retroviruses and possess direct repeats at their ends (long terminal repeats, LTRs).
- PULSED FIELD GEL
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Agarose electrophoresis gel that is run by periodically changing the orientation of the electric field applied to the gel to achieve separation of large fragments of DNA (>20 Kb and up to 10 Mb).
- SYNTENY
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The conservation of the relative order of genes (or of other DNA sequences) in the chromosomes of different species.
- FISH
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(Fluorescence in situ hybridization). A technique in which a fluorescently labelled DNA probe is used to detect and localize a particular sequence on a chromosome with the help of fluorescence microscopy.
- POLYTENE CHROMOSOMES
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A giant chromosome that is formed by many rounds of replication of the DNA. The replicated DNA molecules tightly align side-by-side in parallel register, which creates a non-mitotic chromosome that is visible by light microscopy.
- ANEUPLOID
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Having an unbalanced chromosome number (owing to extra or missing chromosomes). An example is trisomy.
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Meyers, B., Scalabrin, S. & Morgante, M. Mapping and sequencing complex genomes: let's get physical!. Nat Rev Genet 5, 578–588 (2004). https://doi.org/10.1038/nrg1404
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DOI: https://doi.org/10.1038/nrg1404
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