REGULACIÓN DE LAS EMPRESAS DE MEDICINA PREPAGADA

expressed by both of the cell populations. The next step involves separation of the biotinylated double stranded hybrids by the addition of the large protein streptavidin, which binds to biotin. The biotinylated sequences can be separated by phenol- chloroform extraction, in which the sequences bound to avidin are displayed to the interface between the aqueous and organic phases. Non-biotinylated single-stranded cDNAs from the tester, present in the aqueous phase, can then be isolated and inserted into a vector to make a subtracted library. The subtracted library is enriched for both low-abundance and high-abundance mRNAs.

This method is technically difficult, time consuming and unreliable (Wieland et al.,

1990). One limitation is that residual non- induced clones sometimes are present (Sunday, 1995). In general, due to the high complexity of the human genome, subtractive hybridisation techniques do not achieve sufficient enrichment of the sequences that occur only in the tester. This complexity prevents complete

hybridisation. The “target” sequences are enriched only 100 to 1000 times, which is insufficient for more common situations in which the magnitude of enrichment required is 10^ (Lisitsyn et a l, 1993). However, including PCR to allow multiple cycles of selection with limited starting material (Cecchini et a l, 1993) has increased the power of this method. Nevertheless, difficulties still exist with this method. First, the

biotinylation reaction is usually incomplete. Second, the methods used for separating the modified from unmodified molecules are troublesome. As a result of these problems, the subtracted tester is usually contaminated with driver cDNA, which is present initially in excess (Zeng et a l, 1994).

2.1.2.3. Enzymatic degrading subtraction (EDS):

Zeng et al (1994) described a method called EDS for differential library construction and gene cloning. In order to overcome the problems noted with subtractive

Chapter 2_________________________________________________Analysis o f dijferential gene expression

hybridisation library, they developed a single enzymatic method. This method is capable of differentiation between the tester and the driver cDNAs. It also can remove tester-driver heterohybrid and driver-driver homohybrid molecules.

The method involves the incorporation of thionucleotides into the tester rather than biotinylating the driver. This enzymatic modification is more reliable and uniform due to the slow and synchronous excision activity and the high processive activity of Klenow polymerase enzyme. It is more economical since it modifies a small amount of DNA. The most important feature is the removal of the hybrid molecule enzymatically rather than using a physical partitioning method.

2.I.2.4. Representational difference analysis (RDA):

RDA is a method introduced for finding small differences between the sequences of two DNA populations. It is a process of subtraction coupled to amplification. The method builds upon subtractive hybridisation and kinetic enrichment.

In RDA, the DNA complexity of both tester and driver genomes is lowered by preparing a representative portion of each genome a “representation” by six-cutting restriction enzyme digestion. If one restriction fragment is amplifiable (the target) and exists in one representation (the tester) but absent from another (the driver), this target can be enriched by subtractive hybridisation of the tester in the presence of excess of the driver followed by PCR amplification. It has been found that high proportions of the digested fragments do not fall into the amplifiable range, 150-1000 base pairs. As a result, the final representation contains only 2-10% of the total genome (Lisitsyn et a l,

1993). Assuming that sufficiently few sequences are present in cDNA that RDA can be applied without reducing the genome’s complexity, restricted cDNA with a four-cutting enzyme, for instance T>pnl\, would help to generate the representations. This enzyme cutting resulted in a mean cutting length of about 260 base pairs, which ensures that

Chapter 2_________________________________________________Analysis o f differential gene expression

most of cDNA species would have at least one amplifiable fragment. This fragment would be sufficient to identify a gene (Hubank & Schatz, 1994).

RDA was used to clone sequences of two viruses (GBV-A and GBV-B) from the serum of a tamarin infected with the GB hepatitis agent (Simons et a l, 1995), and in the isolation of a genome related to GBV-C from a chimpanzee (Birkenmeyer et a l, 1998). In 1997, TTV was detected using RDA in the serum of a petient with posttransfusion hepatitis unrelated to known hepatitis viruses (Nishizawa et a l, 1997).

2.1.2.5. Differential display

Introduction:

In 1992, Liang & Pardee described a new approach to identify genes expressed differentially in a subset of two or more cell populations. It was called differential display (DD) or according to the PCR nomenclature, DDRT-PCR (Bauer et a l, 1993). The method is based on random primed amplification of a subfraction of mRNA from two cell populations of interest followed by running the amplicons side by side in adjacent lanes on polyacrylamide gels and isolating bands, which are expressed at different levels. The method of DDRT-PCR is illustrated in figure 2.1.

Method strategy:

The method starts with equal quantities of mRNA and uses two distinct types of PCR primers. The first set contains three different anchored 3’ oligo- (dT) primers. These primers generate fragments that originate mostly from the poly (A) tail and extend about 50-600 nucleotides upstream (Liang & Pardee, 1992). The second set contains 5’

random 10-mer primers of arbitrary sequence, previously used for DNA fingerprinting (Welsh & McClelland, 1990). They hybridise with complementary sequences located at varying distances from the 3’ end of each newly synthesised cDNA strand during the ^^S-labelled PCR reaction. The primer pairs are defined to yield -100-200 bands on a 2-

Chapter 2_________________________________________________Analysis o f differential gene expression

3 hours run of a standard sequencing gel. Differential expression is determined by visual inspection of autoradiographs to identify bands present in only a subset of the cell populations. The bands of interest can then be cut out of the dried gels, eluted,

reamplified and utilised as probes for northern blot analyses to confirm the desired differential expression and to provide information about the molecular weight of the identified transcripts. The same cDNA probes, generally between 100 and 500 base pairs in length, can be used to screen directly an appropriate cDNA library, hi addition, the amplified cDNAs can be screened rapidly by sequencing to determine whether the genes are known entities or represent novel genes, whether there are valid open reading frames, and what sequence similarities exist in the nucleotide sequence database

Chapter2 Analysis o f differential gene expression

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DDRT-PCR

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mRNA extraction

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cDNA synthesis with