As mentioned in 4.2-4 the first DNA markers to be discovered were restriction fragment length polymorphisms (RFLP). These were widely used in the 1970's and 1980's and utilise the observation that small variations in DNA sequences can result in differences in the restriction sites of particular enzymes. Most RFLPs are biallelic and therefore not highly informative and their location within the genome is also highly variable. RFLPs were superseded by variable number tandem repeats (VNTRs) or minisatellites. These are short tandem repeat
sequences of DNA in which the number of tandem repeats is variable and this difference can be detected using restriction enzymes. VNTRs are thus more informative than RFLPs but remain labour intensive. The most commonly used markers at present are microsatellites or SSLPs (simple sequence length polymorphisms) which are dinucleotide, trinucleotide or tetranucleotide repeat sequences that can be amplified using the polymerase chain reaction. These markers are highly polymorphic and their informativity is assessed in two ways:
The heterozygosity (MET) of a marker refers to the likelihood that any individual within a population is heterozygous at a specific locus. This is represented by the following formula:
n
MET = 1 - E p)2
1=1
where n= no of alleles, p/is the frequency of the i^^ allele
The polymorphism information content (PIC) refers to the likelihood that the marker genotype of the offspring will allow the accurate determination of the alleles inherited from each parent. By definition the PIC value is always less than the HET.
n n n
PIC = 1 - Z - z Z 2p/2pj2
/=1 /=1 J=i+^
The construction of a high resolution linkage map of microsatellite markers has been crucial to the success of positional cloning projects. The Centre d'Etude de Polymorphisme Humin (CEPH) in Paris has cell lines on 60 three generation families which have been used by the scientific community to type microsatellites to establish a genetic map.
4.3.6
Candidate cloning
Although the specific biochemical abnormality is unknown in many diseases an increasing understanding of molecular biology and biochemistry may identify potential proteins as candidates involved in causing the phenotype. If the genes for these are known they can be investigated for evidence of linkage to the disease phenotype or screened for mutations. In addition animal models may also provide candidate genes for analysis in humans. This approach has been successful in a number of cases.
4.1
The Human Genome project
The human genome project was officially established in the United States on October 1st 1990 following a report by the National Research Council and
many years of discussion within the scientific and political community. Its aim is to genetically and physically map the 60 000 - 70 000 human genes and to sequence the estimated 3 billion nucleotides of the haploid human genome. Robert Sinsheimer, a molecular biologist and chancellor of the Santa Cruz
campus was instrumental in provoking interest in this ambitious biological project and his lead was eventually taken over by the National Institute of Health ( NIH). The head of the Department of Energy's (DOE) Office of Health and
Environmental Research Charles DeLisi had also separately fostered interest in a similar idea and ultimately these two parallel approaches emerged as one to form the National Institute of Health Centre for Human Genome Research headed originally by Dr James Watson. Prominent European scientists were also involved in the conception of the Human Genome Project and it is a truly international collaborative effort coordinated overall by the Human Genome Organisation (HUGO). The founder member of HUGO was Victor McKusick whose contribution to medical genetics is unrivalled. HUGO aims to guide participating countries in the sharing of information and materials and to ensure that the human genome project proceeds rapidly and efficiently.
In parallel to the main aim of the project, funding was also directed to the mapping and sequencing of the genome of model organisms including Yeast, Escherichia coli, Caenorhabditis elegans and the mouse. As Dr James Watson predicted this has contributed significantly to our understanding of the human genome because these organisms have provided insights into some of the basic principles of the molecular machinery of multicellular organisms. A mutant gene identified in a "simple" organism may facilitate the identification of a human gene because of sequence homology identified through the databases. This is the field of comparative biology which allows the evolution of the genome between organisms to be studied.
In the United Kingdom the Human Genome Mapping Project UK Resource Centre (HGMP) at the Sanger centre in Cambridge is funded by the Medical Research Council (MRC). Investigators working in the field of molecular
genetics are able to register with HGMP and this service provides free access to a wide range of information including training courses, genetic databases,
linkage computer programs and molecular genetic materials.
The establishment of the human genome project has acted as a catalyst for the development of improved molecular genetics techniques and the computer software necessary to analyse and disseminate information. It has also encouraged the sharing of information to increase the efficiency with which individual groups produce results. HUGO also has an important role in
establishing the principles and ethics of genetic research and its application to medical genetics.
The projected date of completion for the Human Genome project is September 30th 2005. The project is currently ahead of schedule and a first draught of the complete human sequence is expected by. The complete DNA sequence of model organisms such as fugu and C. elegans is already known.
The challenge for the future has already been referred to as the post genomic era. Identifying and sequencing the entire human genome is just the beginning. Molecular geneticists, biologists and biochemists will then work together to completely elucidate the detailed temporal and spatial regulation, function and interaction of all of the human genes.