4.4.1 Acedb
Acedb is an object-oriented database software with a strong emphasis on graphical maps for navigating in the data. It was written in C by Richard Durbin (The Sanger Centre, Hinxton, UK) and Jean-Thierry Mieg (CNRS, Montpellier, France), and includes a number of features contributed by many users of the system. It was first developed to visually represent physical maps constructed with the contig9 program written by J. Sulston, for the C. elegans mapping project. It is now a general tool for storing genomic and biological information and over 40 known projects are using it to maintain and distribute data. The acedb code and executables for most platforms are available by anonymous ftp from:
ftp.sanger.ac.uk in directory pub/acedb lirmm.lirmm.fr in directory genome/acedb ncbi.nlm.nih.gov in directory repository/acedb.
4.4.2 ORACLE.
ORACLE is a relational database management system available commercially from ORACLE corporation. Redwood City, USA.
CHAPTER THREE: Hybridisation
Fingerprinting of YAC Ciones
1. Introduction
Several methods exist which can reliably demonstrate the overlap between two YAC clones. They can be divided in three classes: hybridisations of labelled probes, STS detection by PCR, and fingerprinting, each very different in their experimental design and in the type of result generated.
The hybridisation of DNA probes to YAC DNA fixed on a solid support followed by the detection of the bound probe, is an extremely robust technique since it relies on the exact homology of long DNA molecules (between a few hundred bp to several kb) with their target YAC DNA. In this sensitive technique, where picograms of DNA can be detected on Southern blots (Southern, 1975), no prior knowledge of DNA sequence is necessary. The yield of the experiment is proportional to the number and diversity of target DNAs screened in parallel and in large scale physical mapping in particular, this latter feature can be exploited by using libraries of large number of clones g ridded at high densities on a solid support (Lehrach et al., 1990). The detection of STSs (Olson et al., 1989) in YAC clones by PCR (Saiki et al., 1988), relies on the enzymatic amplification of a specific and unique sequence from the YAC DNA template. Here, a small stretch of DNA sequence must be determined before two primers can be designed for the PCR reaction. Although this is the most costly and time consuming part of the procedure, it is also this feature which allows a very rapid transfer of information between investigators, since only the primer sequence is needed to reproduce the experiment. Combined with dedicated efforts to generate polymorphic STS markers (Dib et al., 1996) and central repositories of mapping information such as the Genome Database (Fasman et al., 1996), this has established ‘STS content mapping’ of YAC clones as the most widely used technique for establishing genetic and physical maps of the human genome (Chumakov et al., 1995) (Hudson et al., 1995). Both probe hybridisation and STS mapping methods provide a direct evidence for the presence of an overlap between two YAC clones, respectively a specific hybridisation signal or a specific band on an electrophoresis gel.
The fingerprinting method, on the other hand, only indirectly detects overlaps. Mapping by clone fingerprinting is generally based on the separation by electrophoresis of a pool of sub-fragments obtained for each individual clone, followed by the estimation of the different fragment sizes. For each clone, the list of fragment
generating the fragments. Applied to cosmids, it was one of the methods used for mapping the 80 Mb of the Caenorhabditis elegans genome (Coulson et a!., 1986). The size of YAC clones however prevents the use of such restriction digests as a means to obtain fragment pools for each clone insert. Typically between 200 kb and 1.5 Mb, YACs fall within the range of the yeast chromosomes themselves and therefore can not be purified easily. An alternative method is to use Interspersed
Repeat Sequence-PCR (IRS-PCR), where primers are derived from a sequence likely to occur a number of times within a YAC clone. The Alu sequence from the SINE family of repeats was first used for this purpose (Nelson et al., 1989) due to its convenient frequency of approximately once every 4 kb of genomic DNA (Hwu et al., 1986). Two strong disadvantages of fingerprinting methods are first that they depend on a large overlap between clones in order to be reliable, and secondly that the statistical nature of the results necessitates that complementary techniques are used to confirm them. However, it requires very little starting DNA, which can be isolated as a crude preparation of whole yeast genomic DNA, and is very easy and fast to implement (Coffey et al., 1996).
This chapter describes a new method which combines the high degree of sensitivity and reliability associated with hybridisation based experiments, with the speed and ease of implementation of Alu-PCR. The principle is based on the cloning of a pool of Alu-PCR products from the X chromosome, followed by the arraying of the clones at high density on nylon filters, in the form of PCR products. The YAC clones to be fingerprinted are first amplified by Alu-PCR and then hybridised to the filters. Cloned PCR products identified in common between two YAC probes indicate an overlap (Figure 3.1 OA). In addition, the cloned PCR products are readily available as mapping reagents for further experiments. This approach was used to confirm overlaps between YAC clones that were already assembled into clusters in separate experiments (performed by M. Ross), to construct new contigs between previously unclustered YAC clones, and to place the cloned Alu PCR products on the emerging X chromosome YAC contig map, as a reservoir of new markers. Since the technique was to be used to fingerprint several hundred YAC probes by one person, different aspects of the method have been optimised with a view to facilitating the large scale of the experiment, raising the throughput of data production and facilitating the acquisition of the results. A streamlining of the hybridisation procedure was accomplished which included the evaluation of new non-radioactive detection systems and these aspects of the project are described in chapter 4.