Método Analítico y método de Simulación (Monte Carlo) [53]

Using prim ers

Linear am plification

T

D igest w t h M ung Bean N ucleas and PCR

i

Exponential am plification

T

No am plification

Figure 1.14: Schematic diagram o f cDNA RDA procedure (Lisistsyn et at 1993). Step I to 5 are the same in both tester and driver samples but tester cDNA (partial digests) are ligated with a new adaptor to make a new end fo r one o f the samples which is used during the second PCR amplification after hybridisation stage.

Paul, 1997). Consequently, multiple rounds of subtraction are still required. In addition, like all subtractive methodologies, RDA can only compare two populations at a time and has generally requires large (-100 /xg) quantities of starting mRNA.

4.1.4 DNA microarrays

DNA microarray technology is one of the most important recent breakthroughs in experimental molecular biology. It takes advantage of the preferential binding of complementary, single-stranded nucleic acid sequences (Schena et aL, 1995; DeRisi et aL, 1996&1997; Wodicka et aL, 1997). A

microarray is usually a glass slide, to which single-stranded DNA molecules are attached at fixed locations (spots). There may be hundreds or even thousands of spots on an array, each representing a single gene. For display of DETs, RNA is extracted from the sample, which could be for instance a tissue or a cell line treated with a pharmaceutical product, and from control cells. The RNA is then labelled with two fluorescent labels: for instance, a red dye for RNA from the sample population and a green dye for that from the control population. Both labeled RNA extracts are washed over the microarray. Gene sequences from the samples and the control hybridise to their complementary sequences in the spots. The dyes make possible the amount of sample bound to a spot to be measured from the level of fluorescence emitted when it is excited by a light wave. If the RNA from the sample population is predominant, the spot will be red; if the RNA from the control

population is more plentiful, it will be green; if the both RNAs bind equally, the spot will be yellow and when neither binds it will appear black. Thus, the relative expression levels of the genes in the sample and in control populations can be estimated.

This method provides a snapshot of gene activity for thousands of known genes and creates a considerable amount of valuable data. For example, it has been used in diagnosis of cancer by the pattern of gene expression (Alizadeh et aL, 2000; Bems, 2000) and for expression profiling in mammal development (Grimmond et aL, 2000). But it is still relatively expensive, needs specific equipment and is not

useful for the purposes of novel gene discovery. There is a rapid improvement in this technique during the last three years (review, Greenfield, 2000). For example, for a higher resolution view of gene structure. Shoemaker et al. (2001) have applied an ‘exon array’ approach to a detailed analysis of human chromosome 22. They have also used ‘tiling arrays’ to refine the structure of new genes discovered by exon analysis (these techniques will not be described). Recently, accumulation of results using this method has made bioinformatic scientists face the necessity of management and analysis of these huge and increasing volumes of data (Brazma et ai, 2000; Lemkin et al., 2000; see site:

http ://w w w. lech .ncifcrf. go v/M AExplorer/hmaeHelp. html).

4.1.5 Library Screening

In addition to these techniques concerned with comparing patterns of expression, a number of screening strategies have been developed in order to identify potential genes. Some of these are explained below.

4.1.5.1 Random partial sequencing of clones from tissue specific cDNA libraries using vector primers can generate a very large number of expressed

sequence tags (ESTs)(Okubo et al., 1991; Boguski et al., 1993). dbEST is a division of GenBank and isolated ESTs reported from all over the word as part of the

Human Genome Project are available at

http://www.ncbi.nlm.nih.gov/dbEST/dbEST_access.html (So far, more than 6.4 million ESTs from different cDNA libraries and organisms). Each EST has a datasheet which contains sequence data and other information such as tissue source, the name of organism, accession no. and clone name. Non-human ESTs are very helpful to compare with human homologues. ESTs can then be mapped to a chromosome by PCR amplification of somatic cell hybrids (see chapter III, table III.4)(Jones et al 1997) and can be used to retrieve entire genes by using either computational or experimental methods. Gene structure may be revealed from the genomic sequence data using computational programs like GRAIL

(http://www.hgmp.mrc.ac.uk/Registered/Option/xgrail.html) which is used for gene prediction and can quickly identify potential exon/intron boundaries. NIX

(http://www.hgmp.mrc.ac.uk/NIX/index.html) is another computational program that can be used to analyse the obtained sequences by comparing results of many available programs at the same times. Large-scale cDNA sequencing projects have been applied to testicular cDNA libraries (Hoog, 1991; Starborg et aL, 1992; Yuan

et aL, 1995). Following that screening Hoog et aL, (1995) have demonstrated some

differences and similarities in expression patterns between brain and testis by comparing the functional categories of ESTs obtained from the two tissues.

Libraries from different mouse germ cell lines such as spermatocytes have been constructed to search for stage specific gene expression patterns of

spermatogenesis (personal communication, Burgoyne, 1999 and see chapter III, section III.9).

4.1.5.2 Another screening technique is to screen human cDNA libraries at low stringency with non-human cDNA probes that are known (from databases) to be relatively conserved between species. For example, a yeast meiotic gene has been used (serine threonine kinase) as a probe to screen a mouse testis cDNA library (Shalom and Don 1999). In addition, this method could be very useful for finding other members of a gene family. The DAZLA gene was identified by screening a human testis cDNA library with a DAZ probe (Yen et aL, 1996). Screening with genomic clones is technically demanding and variable as some genomic clones, particularly YACs, give a very high background due to their complexity and repetitive sequence content. A converse method which has been used with some success is to screen chromosome specific genomic libraries with pooled cDNAs made from specific tissues (Hochgeschwender et aL, 1989; Ferrari

et aL, 1996).

4.1.5.3 Direct selection of cDNAs from human genomic DNA, is a technique which enables the identification of genes which are expressed in a particular tissue or cell type and which map to a specific chromosome(s) such as Y chromosome (or even to smaller regions of chromosomes) (Lovett et aL, 1991; Parimoo et aL, 1991, personal communication, Makrinou, 2000). The basis of this technique is the hybridisation of a pool of cDNAs to immobilised genomic clones. Non-hybridized sequences are eliminated and selected cDNAs ,i.e., those that

hybridise to ranges of the genomic clones, are recovered by elution and PCR amplification. The PCR products are either cloned or subjected to further

selection/amplification cycles. This technique has also been combined with PCR amplification to facilitate the use of a small amount of mRNA. This method identifies coding sequences which map to large genomic regions in a way which does not depend on number and size of introns or cryptic splice sites. Limitations of this technique may be contamination with mitochondrial cDNAs and repetitive DNA, the long procedure of creating the library and relatively time consuming sequencing of the selected PCR products (personal communication, Makrinou,

2000).

4.1.5.4 Differential screening of a subtracted cDNA library is another method which has been introduced using a combination of this and the array technology, to search for genes preferentially expressed in multiple tissues. For instance, Jin et al. (1997) followed this strategy to identify genes expressed in both testis and ovary. First they generated testis-specific cDNAs using the suppression subtractive hybridisation technique, which were then cloned and arrayed in microplates. The inserts from the subtracted testis-specific library were amplified by PCR and spotted onto filters. The dot blots from 2000 putative clones were screened by hybridisation using either testis- or ovary-specific subtracted cDNA mixtures as probes. The results showed that only three clones were preferentially or exclusively expressed in both testis and ovary. This technique is not simple or without biases and involved a number of stages.

4.1.6 DDRT-PCR

Differential display RT-PCR (DDRT) is a method that has been developed for the identification of genes differentially expressed between different cell or tissue types by creation of an RNA fingerprint for each sample (Liang and Pardee, 1992). Arbitrarily primed PCR fingerprinting of RNA is another very similar method for fingerprinting of RNA populations using an arbitrarily selected primer at low

j^AAAAAAAAAA 1- mRNA (it is possible to use total RNA as well) A A A A A A A A A A A

Arbitrary Primer

26 different 10-mers (A rbitrary prim ers) Sometimes these prim ers are:

an adaptor + 10-nier

5 '— — N N N N N N N N N N 3

IV V T T T T T T T T T T T T 2 - cDNA (cD N A synthesis in 4 specific groups)

JVVAAAAAAAAAAA 3- PCR (am plification at low tem perature)

j^ T T T T T T T T T T T T

spies 1

(Anchored prim ers)

g . y y y y y y ^ y

N = A, C, G and T V = A, C or G

4 Oligo-dTVNs with different N Sometimes these prim ers have

an ad ap to r

^ Specific band