BENEFICIOS TÉCNICOS

_____________________________________AAAAAA cDNA ---

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A ATT______________________ AATT____________________________ AAAAAA

---TTAA TTTTTT - j - A A T T g a m » » I T 4 D N A TTAA ^ ligase % # A A T T --- AATT # # # TTAA--- TTAA Fok 1 Digestion ÜC AT AATT mmm. ■CGTA T T A A I

— S ^ S c G T N (ligation o f one o f 64 ad ap ters)

^ 3 io tin |- | ^ M G C A T _____________________________ AATT ■ ■ ■ C G T N --- ITAA ^ j j j j j j l ^ ^ ^ IXnabeads-strepavidin Dénaturation -NaOH GCAT ■ AATT Prim ers g g g , PCR am plification PAGE

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Figure LI 1-B

OQ n o OQ K) / # S'. do' OQ 00

Figure 1.12: Schematic representation o f the principle o f RLCS and GEF methods. In brief,the first stages, cDNA synthesis with a biotin-oligo(dT) primer and first digestion are the same in both (GEF and RLCS). In GEF method, cDI^As are ligated with an adapter, amplified and then labelled. cDNAs are immobilised using Avidin-Biotin reaction in both techniques. Different digestion stages fo r samples are carried o u t. Differences between samples can be displayed by running on a normal polyacrylamide gel (in GEF) or two dimensional gel electrophoresis (in RLCS). Both techniques represent one message-one displayble fragment feature because only the 3 'end o f each cDNA (mRNA) is immobilised and other parts o f cDNAs resulted fro m digestion are washed away. D Z >

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4.1.2 Subtractive hybridisation and suppression subtractive hybridisation (SSH)

An approach to the molecular analysis of differential gene expression is to construct cDNA libraries corresponding to different tissues or developmental stages, and then to enrich for genes expressed in a particular tissue or at a particular time by subtractive hybridisation. Screening involves checking

sequences (clones), picked at random from two libraries, for presence in one of the original RNA samples in higher concentration than in another. Subtractive

hybridisation is based on a procedure that increases the effective concentration of induced sequences expressed in an experimental RNA population (target) but not in a control RNA population (driver) (Sargent, 1987). This technique enriches samples for DETs prior to differential screening. Unhybridised cDNAs, which represent differentially expressed, tissue specific sequences can thus be isolated (Kara et aL, 1991). However, during subtractive hybridisation and cloning

protocols for the isolation of activity-dependent genes, two main points have to be considered which are of general concern for a variety of other applications. This method requires a large amount of mRNA and is not particularly effective in identifying low abundance transcripts, and also following subtractive

hybridisation an enrichment of induced sequences is frequently accompanied by an accumulation of sequences unable to form heteroduplexes for purely technical reasons ( Li, et a/.,1994; Konietzko and Kuhl, 1998). However, some

contemporary techniques of subtractive hybridisation largely overcome the problem of bias toward abundant mRNAs (Gurskaya et aL, 1996; von Stein et aL,

1997). By using a combination of restriction enzyme digestion, hybridisation and PCR amplification a version of the subtractive hybridisation technique has been developed and is termed suppression subtractive hybridisation (SSH). This PCR- based cDNA subtraction method is used to amplify target cDNA fragments (differentially expressed) but simultaneously suppresses nontarget DNA amplification. The procedure, which has been explained by Diatchenko et aL,

1- cDNA synthesis from two mRNA samples under com parison (tester and driver)

2- Both cDNAs are separately digested to obtain shorter, biunt-ended DNA 3- Two tester populations are created with different adapters but not driver cDNA

4- First hybridisation lead to equalisation and enrichm ent o f differentially

expressed sequences

Tester c D N A with adaptor 1

Driver cD N A (in excess) Tester cD N A w ith adaptor 2

First

5- Second hybridisation generate tem plates for PCR am plification

6- During the first PCR am plification , using suppression PCR, only

differentially expressed sequences are amplified exponentially

hybridization

»

Second hylrridization

Fill in the 3' ends by D N A polym erase in the first PCR cycle

First PCR using H & n

no am plification

linear am plification

not successfully am plified (

exponential amplification

), suppression PCR (Siebert et al., 1995)

7- Second PCR am plification using K , to enrich differentially expressed sequences

Figure 1.13: Scheme o f the SSH technique. Adapters are ligated to digested ds cDNAs made from tester and driver mRISAs. Green (with and without a black dot), black and blue boxes are adapters (which can be used as a site fo r nested primers) and two different primers. PCR products would be displayed on a gel and cloned in a set o f vectors fo r

to make one of these a mixture of RNA from several tissues, thus comparing more than one tissue at a time which can be an a significant advantage of this technique.

4.1.3 Representational difference analysis (RDA)

Representational difference analysis (RDA) is another approach to the

identification of the differences between complex genomes and could be valuable as the first step for the positional cloning of genes of interest (Lisitsyn et aL, 1993; Hubank and Schatz 1994). RDA is basically a method of DNA subtraction, to investigate differences at the DNA level rather than RNA, similar to other methods such as chromosome painting, comparative genomic hybridisation (CGH),

restriction landmark genome scanning (RLGS) and high-throughput analysis of LOH (Loss of heterozygosity). It can be used in situations such as the comparison of normal and tumour tissue (review. Gray and Collins 2000). Numerous studies have been done in cancers using RDA for the detection of genetic lesions, such as deletion (reviews, Baldocchi and Flaherty 1997; Wallrapp et aL, 1999; Frohme et aL, 2000). A possible application of RDA might be to display differences between normal men and patients with infertility caused by deletion with comparison of genomic DNA of fertile and infertile males. RDA has been successfully adapted to identify genes that are differentially expressed between two populations of cells. Figure 1.14 shows the steps involved in the RDA protocol. Representative cDNA fragments from the two population to be compared are first generated by restriction endonuclease digestion of cDNAs followed by PCR amplification. The resulting mixtures, termed “amplicons”, are then subject to successive rounds of subtractive cross-hybridisation followed by differential PCR amplification. This leads to progressive enrichment of cDNA fragments that are more abundant in one

population than the other. However, absolute expression level has not been a crucial determinant for identifying genes, because fragments from both abundant and rare transcripts have been isolated (Braun et aL, 1995). As a disadvantage, this method does not resolve the problem of the wide differences in abundance of individual mRNA species because each RDA recovers typically only 6-12 differentially expressed cDNA fragments (Hubank and Schatz 1994; Braun et aL, 1995; Chu and

mRNA From Tester and Driver 1- cDNA synthesis 2- Digestion .A A A A 3- Ligation 4 PCR amplification cD N A synthesis .A A A A ■TTTT

Restriction digest using 4 cutter enzym e

.A A A A T T T T

A daptor Ligation

Fill in 3 'en d (in the first cycle o f PCR)

PCR using the adaptor primer

Tester Driver J 5- Digest 5- Digest 6- Ligate (new adaptor'

7- Mix (1:100), D enature, H ybridise

Driver: Driver Tester: Driver Tester: Tester

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