Cantidad de litros de leche/vaca

Contig assembly relies upon identifying overlapping clones across a region by linking them with one or more reference points, and bridging gaps by isolating new clones that extend further. The strategy depends on the density of available markers within the region and the size and complexity of the library screened. Genetic markers serve as anchors for identifying seed YACs as starting material for the development of large cloned contigs. These markers also provide positional information for the order and orientation of other STSs which are placed on the YAC contig and is a means to integrate physical with genetic maps. To save time-consuming screening for clones, it is beneficial to exhaust all the resources available to the scientific community. YACs can be identified which have already been mapped to the region of interest and have been placed on an integrated map (Cohen et a l 1993, Chumakov et a l 1995).

However, contig assembly is rarely completed with these clones and it is usually necessary to augment the map with other clones by screening the publicly available libraries and to facilitate the ordering o f markers across the region.

One approach to determine overlaps between clones, which was employed here, is STS content mapping (Kere et a l 1992). This is where PCR or hybridisation is

used to determine the presence of unique markers or probes in clones. Overlapping clones are identified when an STS is present in both o f them, and the order o f STS markers can be determined by deciphering a pattern from which clones they accommodate. The PCR-based approach is more favourable as it is faster and only the primer sets have to be unique and specific. In hybridisation based mapping, if a probe includes repeats such as microsatellites, it can hinder analysis. The STSs on the other

Physical Mapping o f the RP9 Critical Region

hand can be polymorphic probes, cDNA probes or others generated from sequences in the region and must be site specific. At the start of contig construction in the RP9

region the density of STSs were low, mostly including poly (CA)n markers which had

been ordered by genetic analysis.

A second approach to determine overlaps between clones is by fingerprint analysis. This method is usually performed in probe-poor regions or when handling a large number o f clones. Unpurified YAC DNA can be digested with one or more restriction enzymes, then separated by Pulse Field Gel Electrophoresis (PFGE), and common bands are identified by Southern blot analysis using a human specific repeat probe (e.g. Alu or LINE-1 DNA) (Bellanne-Chantelot et a l 1992). A more rapid alternative is to generate a fingerprint by 'miox-Alu PCR (Nelson et a l 1989). Alu repeats represent the major family of short interspersed repeats (SINE) in mammalian genomes and are spaced at an average of one every 3-6 kb (Britten et a l 1988, Moyzis

et a l 1989). Human specific oligonucleotides designed from the terminal ends of the

repeat and which direct away from it, are^sed to amplify inter-^/w products in the YAC clones, providing the distance between the adjacent repeats are amenable to PCR. The samples are fractionated by gel electrophoresis, producing a fingerprint consisting of PCR products of characteristic length. The orientation of a proportion of the repeats are inverted, which therefore allows the use of single primers to produce different fingerprints. Inter-^/w PCR products can also be used as a source for the production of additional probes or STSs from less well characterised regions of the genome (Nelson et a l 1991 and Cole era/. 1991).

In cases where there is an insufficient number o f probes to assemble complete contigs directly, terminal sequences from a clone can be isolated and used as a probe to screen a library for the identification and isolation of overlapping clones. This approach, termed chromosome walking, is usually employed to bridge gaps between anchored clones that are physically separated. The walk is usually performed bi- directionally unless the orientation of the clone is known. Many methods for the isolation o f end sequences have been reported. The recovered end clone products can be used directly as probes, or are more commonly sequenced to generate PCR-based STSs to isolate overlapping YACs.

Physical M apping o f the RP9 Critical Region

BACs, PACs and cosmids can be incorporated into the contig at a later stage, which can be used for resolving STS order and to provide a more manageable and stable source for further manipulations. The size of individual YACs can be determined by Pulse Field Gel Electrophoresis (PFGE) (Schwartz and Cantor 1984). This method can be used in a range of further manipulations to characterise YAC clones.

3.1.5 Aims.

At the commencement of this study, the RP9 disease interval had been defined by the flanking markers D7S484 and D7S526 (Ingleheam et al. 1994), both of which exhibited one crossover with the disease allele from the affected members of the adRP7 family. The construction of a complete contig consisting of ordered overlapping clones was the next stage in the positional cloning o f the disease gene. This study describes contig assembly in the distal part of the region. The first step was to order and assign YACs with STSs to create a contig. Secondly, known genes and ESTs were mapped and ordered onto the contig to assemble a transcriptional map and identify any genes that may be candidates for RP9. The contig would also provide an estimation of the physical distance of the region. Most importantly it would provide a valuable resource to identify genes residing in this area.

3.2

Results

Contig assembly was a continuous process and as new PCR-based markers or STSs were assigned to the region, they were incorporated into the existing map. New YAC clones were isolated to increase clone redundancy at critical points and to help resolve the STS order. Both Alu fingerprinting and STS content mapping strategies

were applied, as they provide a rapid method of determining overlaps between clones. The results are presented as a non-chronological summary o f the work. The resulting contig provided a useful resource both to compare the genetic and physical distances and as a foundation for a transcript map of the 7pl4-15 region.

Physical M apping o f the RP9 Critical Region