Recipientes

After a gene has been assigned to a relatively small area on a chromosome through genetic linkage maps, before moving on to the characterisation o f transcripts in the region, this region must be recovered in cloned DNA to create a resource from which to isolate candidate transcripts. Physical maps o f DNA can have several levels o f detail, from the banding patterns o f the chromosomes, to clones o f overlapping segments o f DNA, and ultimately to the base-by-base sequence o f DNA (Figure 1.13) reflecting different levels o f resolution.

1.5.1 Low resolution physical mapping

The low-resolution physical maps are achieved commonly by cytogenetic methods, and the smallest map unit that can be resolved is traditionally one to several megabases of DNA. However, with the recent development of various technologies the level o f resolution has increased to a maximum of 10-20 Kb.

1.5.1.1 Somatic cell hybrid

Somatic cell hybrids can be generated, under certain experimental conditions, inducing the fusion o f cultured cells from different species. In human genome mapping, human and mouse or hamster hybrids are commonly used (Ruddle 1981). Due to the unstable nature o f hybrid cell lines, human chromosomes tend to be randomly lost, thus generating a panel o f hybrids each containing the full set of rodent chromosomes plus a few human chromosomes. Hybrids can be selected for retention o f a given human chromosome if it corrects an otherwise lethal abnormality in the rodent cell.

The human chromosome in the somatic cell hybrid can be identified upon hybridisation or PCR using human specific sequences. More refined mapping can be achieved by using hybrids containing parts of human chromosomes (such as those generated from human cells that have a chromosomal translocation or deletion).

1.5.1.1.1 Radiation hybrid mapping

A major development in somatic cell genetics has been the introduction o f radiation hybrids (RH) to generate physical maps (Walter et a l 1994). RH are constructed by first

F ig u r e 1 .13: Multiple levels of human chromosome mapping. The lowest resolution is provided by cytogenetic maps, and then increases through progressively more detailed physical maps, to achieve its maximum with the base-by-base sequence o f DNA. (Picture taken from Access Excellence, http://www.accessexcellence.org/)

Chromosome

Physical Map

Large Clones

Physical Map / ;

Small Clones '

^

DNA^ï?!

Sequence

^mmS:

using X-rays to break the chromosomes o f a human cell into several fragments whose sizes are a function o f the dose o f radiation. The irradiated cells are then fused with the rodent cells, each o f which will then harbour a complete set o f rodent chromosomes and a random set o f human chromosome fragments. When a set o f DNA markers is assayed in a panel of such radiation hybrids, the pattern of cross-reactivity can be used to construct a map, by taking advantage o f a principle similar to that used in genetic linkage analysis, the nearer together the two DNA sequences are on a chromosome, the lower the probability that they will be separated by a break produced by the X-ray irradiation. Distances between markers are thus calculated and measured in centiRays (cR), which is a function o f the dosage o f radiation.

The range o f resolution o f RH mapping can be varied by altering the X-ray dose used to fragment the chromosomes.

1.5.1.2 In situ hybridisation

Chromosomal in situ hybridisation is a powerful technology to assign chromosomal location and relative order o f DNA sequences to chromosomal sub-regions. Chromosomes in metaphase on a microscope slide are hybridised with a suitable probe isotopically-labelled. More recently, the sensitivity and resolution o f in situ hybridisation has been largely increased by the development of fluorescent in situ hybridisation (FISH), which uses fluorescently-labelled probes that can be easily scored by eye under a fluorescence microscope (Trask 1991).

The resolution o f conventional FISH on metaphase chromosomes is 1-2 Mb (Trask et al. 1991). The use o f the more extended prometaphase chromosomes can permit a higher resolution (30 Kb-1 Mb). More recently, a technique has been developed, which mechanically stretches out free chromatin fibres from interphase nuclei on a microscope slide, this can achieve a resolution of 10-20 Kb (Haaf and Ward 1994).

1.5.2 High resolution physical mapping

The high-resolution physical maps, resolved through molecular methods, have a resolution that can go from hundreds o f kilobases to a single nucleotide.

1.5.2.1 Long-range restriction mapping

Long-range restriction mapping enables the determination of the order and distance of genetic markers by Southern blot analysis o f complete or partially digested genomic DNA. Two markers are physically linked if they co-hybridise to the same band and their maximum distance is indicated by the smallest restriction fragment shared by both probes. The resolution o f restriction mapping depends on the frequency of the recognition site of the restriction enzyme used. This method can be applied to intact YAC, PAG or BAG cloned contigs, enabling identification o f overlaps, rearrangements, deletions, translocation and chimerism that may be present.

The development of pulsed field gel electrophoresis (PFGE, Schwartz and Cantor 1984) has made restriction mapping possible, owing to its ability of separating large restriction fragments, which could not have been achieved by standard gel electrophoresis. Whilst standard gel electrophoresis allows separation o f fragments o f up to 50 Kb, PFGE allows the resolution of fragments as large as 10 Mb, This is achieved by introducing electric fields that change strength or direction over time, thus forcing the large DNA molecules to periodically reorientate in new directions. The time taken for a DNA molecule to reorient under the new electric field is strictly size-dependent, allowing very large molecules to be efficiently fractionated.

1.5.2.2 Clone contig assembly

The construction of the ultimate physical map o f a region of interest requires the assembly o f cloned DNA fragments, which collectively provide full representation of the given region. To ensure a complete coverage, the clones should contain overlapping inserts forming a continuous tiling path or contig. The strategy involves partial digestion of the genomic DNA and cloning of the fragments generated so that DNA sequences of each clone partially overlap with at least some other clones in the library.

One o f the main approaches to determine the overlaps between clone inserts and to assess this overlap is by sequence tagged site (STS) content mapping (Kere et al. 1992). The strategy is based on PGR screening o f STS markers in different clones, overlapping inserts are therefore identified when an STS is present in both. Based on the assumption that an STS

is unique to a particular genomic locus, all clones containing that STS must have originated from that same locus and must therefore overlap.

An alternative approach for the identification of overlapping clones is by fingerprint analysis. This method can be performed without first separating the clone insert from the host chromosomal background, by digestion o f clones, separation by PFGE and identification o f common bands by Southern analysis o f clone inserts using human-specific repetitive sequence probes (e.g. Alu, Wada et al. 1990, or LINE-1, Bellane-Chantellot et al. 1992). A more rapid alternative to generate a fingerprint is to perform mitv-Alu PCR (Nelson et al.

1989) using primers designed from the terminal end o f the repeats, and compare the pattern o f products generated by electrophoresis.

To bridge gaps in existing contigs or to extend outwards from both ends o f a contig the procedure generally employed is chromosome walking. In this strategy, terminal sequences o f a clone are used as a probe to screen a library for the identification and isolation o f overlapping clones. Different methods have been developed for the isolation o f clone ends, most of which are PCR-based (e.g. inverse PCR, Ochman et al. 1988, vectorette PCR, Riley et al. 1990, vector-yl/w PCR, Nelson et al. 1991).

1.5.2.2.1 Yeast artificial chromosomes versus bacterial host systems

The development o f YACs, which contain inserts o f up to 2 Mb (Burke et al. 1987, Larin et al. 1991), has greatly increased the genomic cloning capacity over cosmids, which contain inserts o f approximately 40 Kb. Owing to their large size, contigs o f up to several megabases can be assembled over large regions of chromosomes. They can often provide cloned intact large genes, also permitting analysis o f long distance regulatory elements, generally absent from cosmid constructs.

The downside o f YACs is that they often contain chimeric inserts, which are DNA fragments containing two or more non-contiguous regions o f the genome. This may be due to co-ligation o f two different restriction fragments prior to transformation. Unstable YACs that can easily lose the insert or that have the tendency to delete internal regions from their inserts are also common, posing another disadvantage in the use o f these clones. For the fme- structure analysis that must follow a long-range physical map, alternative clones based on bacterial host systems have been developed along the more traditional cosmid vectors. These

second generation clones, bacterial artificial chromosomes (BACs) and PI artificial chromosomes (PACs), have insert sizes ranging from 100 to 300 Kb (Monaco and Larin 1994). While the insert size is much smaller than that o f YACs, their greater stability makes them very useful in positional cloning projects as they provide a more faithful representation o f the original DNA, and they exist in many copies per cell, providing a high yield.

The easy availability of YAC libraries and the large insert capacity o f the clones provide a very useful starting point to cover large disease regions and YACs have become the initial tool o f choice for cloning contiguous large regions o f DNA. At a later stage in contig assembly, bacterial host systems can also be incorporated to provide a more manageable and stable resource for further manipulations and for developing high-resolution physical maps.