ENERGÍA EÓLICA [16] [1]

The study of genes and their proteins provides information about cellular growth, communication, and organisation and aids the understanding of the complex biological signals and pathways within each cell. In the past decade, the genomes of species including bacteria, yeast. Drosophila, Arabidopsis thaliana and the nematode C. elegans have been sequenced. Many more complete genome sequences are soon to be available and the goal of the Human Genome Project is to sequence the entire human genome. The exponential accumulation of human genome raw sequences has emphasised the need to be able to identify expressed genes within repetitive and non-coding sequences especially those genes related to clinical disorders.

Recently, several protocols have been developed to access genes with particular expression profiles thus identifying differentially expressed transcripts (DETs). In this section the principles, advantages and disadvantages of some of the commonly used techniques for identification of genes and DETs are reviewed.

4.1.1 RNA fingerprinting methods for DETs

The reasons for identifying DETs may be to compare transcripts expressed in different tissues and find those unique to one tissue, to compare transcripts

expressed in normal and disease tissue, or to find sequences in common between specific tissues such as tumour tissues which are not in normal tissues. One way to do this is to generate a “fingerprint” of the transcripts by amplifying them by PCR and then separating the fragments by electrophoresis and comparing the band patterns that are generated.

There are a number of protocols which employ variations on this principle. Some use randomly generated (arbitrary) PCR primers whereas others first cleave

cDNA fragments with restriction enzymes and then anneal linkers to the overhangs (Vos et al, 1995; Ivanova et a l, 1995; Kato, 1996).

For example, a technique for DNA and cDNA fingerprinting named amplified fragment length polymorphisms (AFLP), which is used to detect polymorphism, should be mentioned for this application (Vos et aL, 1995; Money et aL, 1996). In this method, the same principle of fingerprint production is used: a pool of

restriction fragments ligated to adapters is amplified with adapter-specific primers extended by several bases. This may reveal polymorphism when applied to genomic DNA. AFLP produces very good and reproducible fingerprints, but cannot be used for efficient display of cDNA fragments because it lacks the “one messenger-one displayable fragment” feature. In fact, in AFLP there is a mixture of cDNA

fragments several times more complex than the original mRNA pool because each messenger is represented by several independent restriction fragments. In contrast, as explained later in other techniques a cDNA is digested but only the 3'end fragment is displayed.

Two other methods which rely on amplification with an adapter-oligo(dT) primer and adapter-specific primer after processes of digestion and ligation have been introduced. These methods are for comparison of gene expression profiles and schematics illustrating the processes of ordered differential display (ODD) (Matz et aL, 1997; Matz and Lukyanov 1998) and another similar technique suggested by Kato (1996) are both shown in figure 1.11.

Two similar technique are Gene Expression Fingerprinting (GEF) and Restriction Landmark cDNA Scanning (RLCS)(lvanova and Belyavsky, 1995; Suzuki et aL, 1996). As shown in figure 1.12 the techniques are rather complicated and additionally RLCS employs two-dimensional gel electrophoresis which requires a lot of skill to obtain reproducible patterns. These techniques will not be described in detail.

Figure L U : Outline o f two different techniques which are based on using restriction enzyme digestion and ligation o f primer-adaprors fo r identifying DETs, Schematic representation o f the ODD protocol (right) and another similar method which have been introduced by Matz et al.(1997) and Kato (1996), respectively.

A ) In the ODD method, after cDNA synthesis, using an oligo(dT) prim er which has a small adapter sequence at 5 ' end, the cDNA samples are digested by R sal. They are taken fo r ligation with pseudo-double-stranded adapter, a long oligo and a short one, complementary to the 3 'part o f the longer one. PCR amplification is carried out using adapter-specific primer and the oligo(dT) primer. During the firs t PCR, fragm ents flanked by inverted terminal repeats can not be amplified with the single prim er corresponding to the outer part o f the repeat, based on PCR suppression effect (Siebert et al., 1995;

Lukyanov et al., 1996). The second PCR at the high annealing temperature (67°C) using both the oligo(dT) and a labelled adapter-specific primer extended by two arbitrary, but defined, bases at their 3" ends produces a subset o f PCR products corresponding 1/192 part o f the total pool (as there exist 16 possible variants o f adapter-specific prim er extension and 12 o f the oligo(dT) prim eras shown in the figure). In addition, an intentional

mismatch (with respect to the sequence o f adapter-restriction site junction) has been made in the sequence o f the extended adapter-specific primer in the fou rth position from its 3 'end. It has been fo u n d that this enhances the distinction substantially, because in such a situation a perfect match to all three 3 ' distal bases (including the two "selector' bases corresponding to extension) is essential fo r efficient priming. As a result, the population o f 3 'fragments is subdivided into 192 subsets, which are displayed on an ordinary sequencing gel.

B ) In the first step o f Kato ^s protocol, cDNA is digested by EcoRI and ligated with an EcoRl adaptor and in the second stage cDNA is digested by a class IIS restriction enzyme (such as Fokl) which produces fo u r base overhangs o f unknown sequence. Then, the cDNA is ligated separately to 64 adapters, each designed fo r one o f the possible

overhangs. The adapters have a 5 'fo u r nucleotids overhang o f which the outmost is N (a mixture o f A, C, G and T) and the other three are selective defined bases. The difference occurs at the ligation step, as the ligase attaches a specific adaptor almost exclusively to the complementary overhangs. The ligation products are recovered on streptavidin dynabeads using biotin attached to the adapter, and the resulting subset is amplified from the

bound sample with the adaptor-specific prim er and one o f the three 1 base anchored T primers. By repeating the experiment with three different class IIS restriction enzymes,

the cDNA can be subdivided into 576populations.

In the ODD method, in obtained samples each transcript is represented by a single

fragm ent o f characteristic length, between oligo(dT) prim er and adapter sequence but not in the original Kato'sprotocol. In the Kato^sprotocol by using oligo(dT)primer with an adapter during cDNA synthesis and deletion o f the first step digestion it can be achieved the **one messenger-one displayable fragm ent” feature.