Basic tools of Molecular analysis

The earliest genetic methods studied morphological appearances of chromosomes associated with inherited disease. Human chromosomes are most conveniently studied in mitogen-stimulated peripheral blood lymphocytes in which cell division is arrested in metaphase when the chromosom are condensed. The cells are swollen with a hypotonic solution spread on microscope slides to separate the individual chromosomes and stained.

A classical favourite dye is Giemsa which preferentially binds to A-T base pairs resulting in the G-banded appearances visible in the metaphase spread . More recently, methods using fluorescent probes have been used (fluorescence in situ hybridisation, FISH), particularly to identify chromosomal translocations and locate the site of new genes. With the advent of chromosomal “painting', where colour probes specific to each chromosome are used, it is likely that this will become a more routine method of analysis.

DNA can be obtained readily from any nucleated cell by breaking open the cell with proteinases and detergents, removing non-DNA components using specific enzymes or lipid solvents such as phenol, and precipitating the DNA.

When suspended in solution, pure DNA is colourless and invisible, although the presence and concentration of DNA can be deduced by the absorption of light at a wavelength of 260 nm. However, it is usually more convenient to view DNA directly. Some methods are non-specific, such as ethidium bromide labelling. Most exploit the specific base-pair binding discussed on page 3 by use of a short stretch of DNA which is single-stranded and complementary to the DNA sequence under analysis. Such short singlestranded DNA molecules are referred to as 'probes' and are usually labelled to emit ionising radiation or fluorescent light.

Analysis of naturally occurring DNA is made difficult because DNA consists of a series of large molecules which are difficult to handle, and each cell provides only two copies of the sequence of interest in 6 x 10 range to power 9 base pairs.

To facilitate handling, DNA can be cut into smaller fragments using restriction endonucleases . These enzymes cleave double-stranded DNA at a specific recognition site usually made up of a four or six base-pair sequence. Any change in the recognition site abolishes the ability of the restriction enzyme to cut the DNA. As these cleavage sites are specific, characteristic restriction fragment' sizes should result. This would still be of little benefit were it not for the fact that fragments of DNA can be separated easily. Since DNA is negatively charged, it will move towards the anode in an electric field, and as increased DNA mass will impede movement through gel solids such as agarose or acrylamide, during gel electrophoresis smaller pieces of DNA migrate more rapidly and the DNA fragments separate according to size Gel electrophoresis is also used to separate DNA fragments generated by methods other than restriction enzyme digestion.

Nevertheless, detection of any DNA sequence of interest can be compared to finding a needle in a haystack. Labelling the sequence in question with a probe provides a valuable means of detection. This may involve the process of Southern blotting, whereby after the DNA is size-separated on a gel, the gel is dipped briefly in strong alkali to render the DNA single-stranded and the DNA fragments are then transferred by blotting to a nitrocellulose or nylon filter such that their relative positions remain unchanged. The DNA-containing filter is then immersed in a solution containing a probe which hybridises only to complementary sequences. After washing the unbound probe off the filter, the filter is exposed to X-ray film, and the position of the fragment(s) in question will be revealed by dark bands on the film, known as an autoradiogram .

The analyses described above do reveal a tremendous amount of genetic information, but they remain limited by the small number of available DNA molecules. The following sectio describes the two main methods to amplify the speo sequence of DNA in which the investigator is interested.

Microorganisms can take up exogenous DNA by a process known as transformation, but unless this DNA is incorporated into the host genome, it will tend to be lost during the process of cell division. However, the same microorganisms can also take up DNA which has been digested with restriction enzymes and incorporated (by joining together sticky ends) into a self-replicating unit, or vector. In this case, the vector and the exogenous DNA which it contains will be replicated within each cell, and bacterial cell division will result in large numbers of infected cells from which the vector-associated DNA can be prepared separately to the host DNA. Every cell derived from the founder cell should contain the same vector DNA, providing a means to amplify and purify DNA. In routine genetic manipulation experiments, the host microorganism is usually E. coli bacteria, and the vector an infecting virus (bacteriophage), or a plasmid analogous to mitochondria. Other synthetic vectors such as yeast artificial chromosomes (YACs) have been developed to allow host bacteria to propagate larger stretches of exogenous DNA and are particularly important in gene mapping.

The development of polymerase chain reaction technique in the mid-1980s revolutionised molecular biology, and again is based upon the base-pairing. It allows specific amplification of up to 1010 copies of a particular stretch of DNA from a single DNA template. The essence is very simple. Short stretches (around 20-25 nucleotides) of single-stranded DNA are added to the template DNA. These are complementary to the sequence to be amplified, and under the correct conditions, will anneal to single-stranded template DNA and 'prime' DNA replication. The products from each round of replication can serve as the template for the next cycle of amplification and, over a number of cycles, exponential amplification ensues.

Molecular methods allow the deciphering of the genetic code by determining the sequence of bases along a stretch of prepared DNA. Sequencing methods are based on the DNA synthetic process, either carried out in a single synthetic step or in PCR-based cycles. Many of these methods exploit the ability of dideoxynucleotide triphosphates (ddNTPs) to prevent elongation of the newly synthesised strand if they are incorporated at the 3' end to the daughter strand of DNA instead of the relevant deoxynucleotide triphosphate (dNTP) . The products from four separate reactions, each containing all four dNTPs but a different ddNTP, are size-separated on a gel in separate tracks. As the fragments migrate proportional to their length, a ladder pattern is generated in each lane in which each band represents a specific termination event, and the sequence can be read from the shortest fragment at intervals of one nucleotide across all four lanes. Again, this process can be highly automated, with sequences read by computer rather than the eye.

The Human Genome Project (HGP) is an international programme of research which aims to determine the sequence and structure of all functional human genes (and important microorganisms). Although driven by molecular geneticists with an aim to improving our fundamental knowledge of biological processes, the HGP has enormous implications for all types of human disease. The biological questions which will be addressed should improve our therapeutic options, and the methods used to assist the HGP's sequencing efforts have generated resources and sequence which are facilitating the detection of abnormal sequences associated with disease states. However, a major issue for science and medicine to address is what to do with this information. The identification of a genetic susceptibility state in an individual may allow the institution of preventive and therapeutic measures to slow or prevent the development of a disease, but such information carries huge implications for individuals, ranging from the psychological impact of knowing that they are likely to develop a particular disease, to interference with their position in society-for example, in terms of health insurance. Similarly, knowing that some individuals are relatively resistant to disease (e.g. HIV infection) may hamper efforts to improve society's response to a major health risk. Medicine and society have not yet formulated safeguards to ensure that the recent molecular advances result in predominantly beneficial results.