Utilización de lipasas para la síntesis de TAGs estructurados

The probe-restriction enzyme combina-tions may identify two or more differently sized fragments. Polymorphism is revealed whenever the recognized fragments are of non-identical lengths.

Differences in size of restriction frag-ments are due to: (i) base pair changes that result in gain and loss of restriction sites;

and (ii) insertions/deletions at the restric-tion sites within the restricrestric-tion fragments on which the probe sequence is located.

Molecular probes are DNA fragments isolated and individualized by cloning or PCR amplification. They may originate from fragmented total genomic DNA and thus contain coding or non-coding sequences, unique or repeated, of nuclear or cytoplas-mic origin. They may also be complementary

DNA (cDNA). The standard procedure for developing genomic DNA probes is to digest total DNA with a methylation-sensitive enzyme (e.g. PstI), thereby enriching the library for single-copy sequences (Burr et al., 1988). Typically, the digested DNA is size fractionated on a preparative agarose gel.

DNA fragments ranging from 500 to 2000 bp are excised and eluted for cloning into a plasmid vector (e.g. pUC18). Digests of the plasmids are screened for inserts and their lengths can be estimated. Southern blots of the inserts can be probed with total sheared genomic DNA to select clones that hybrid-ize to single- and low-copy sequences and to eliminate clones that hybridize to medium- and high-copy repeated sequences. Single- and low-copy probes are screened for RFLPs among a sample of genotypes using genomic DNAs digested with restriction endonucle-ases (one per assay). Typically, in species with moderate to high polymorphism rates, two to four restriction endonucleases with hexanucleotide recognition sites are tested.

EcoRI, EcoRV and HindIII are widely used.

In species with low polymorphism rates, additional restriction endonucleases can be tested to increase the chance of find-ing a polymorphism. Both the theory and the techniques for RFLP analysis in plant genome mapping have been intensively reviewed (Botstein et al., 1980; Tanksley et al., 1988).

Most RFLP markers are co-dominant and locus specific. RFLP genotyping is highly reproducible and the methodology is sim-ple and requires no special instrumenta-tion. High-throughput markers (e.g. cleaved amplified polymorphic sequence (CAPS) or insertion/deletion (indel) markers) can be developed from RFLP probe sequences.

The CAPS technique, also known as PCR-RFLP, consists of digesting a PCR-amplified fragment with one or several restriction enzymes, and detecting the polymorphism by the presence/absence restriction sites (Konieczny and Ausubel, 1993).

RFLP markers are powerful tools for comparative and synteny mapping.

However, RFLP analysis requires large amounts of high quality DNA and has low genotyping throughput and is very diffi-cult to automate. Most genotyping involves radioactive methods so its use is limited to specific laboratories. RFLP probes must be physically maintained and it is therefore difficult to share them between laboratories.

In addition, the level of RFLP is relatively low and selection for polymorphic parental lines is a limiting step in the development of a complete RFLP map.

RAPD

Williams et al. (1990) and Welsh and McClelland (1990) independently described the utilization of a single, random-sequence oligonucleotide primer in a low stringency PCR (35–45°C) for the simultaneous ampli-fication of several discrete DNA fragments referred to as random amplified polymor-phic DNA (RAPD) and arbitrary primed PCR (AP-PCR), respectively. Another related technique is DNA amplification fingerprint-ing (DAF) (Caetano-Anollés et al., 1991).

These methods differ from one another in primer length, the stringency of the con-ditions and the method of separation and detection of the fragments. They all can be used to identify RAPD.

The principle of RAPD consists of a PCR on the DNA of the individual under study using a short primer, usually ten nucleotides, of arbitrary sequence. The primer which binds to many different loci

is used to amplify random sequences from a complex DNA template that is comple-mentary to it (or includes a limited number of mismatches). This means that the ampli-fied fragments generated by PCR depend on the length and size of both the primer and the target genome. Ten-base oligomers of varying GC content (ranging from 40 to 100%) are usually used. If two hybridiza-tion sites are similar to one another (at least 3000 bp) and in opposite directions, that is, in a configuration that will allow the PCR, amplification will take place. The amplified products (of up to 3.0 kb) are usually sepa-rated on agarose gels and visualized using ethidium bromide staining. The use of a single 10-mer oligonucleotide promotes the generation of several discrete DNA products and these are considered to originate from different genetic loci. Polymorphisms result from mutations or rearrangements either at or between the primer binding sites and are visible in conventional agarose gel electro-phoresis as the presence or absence of a par-ticular RAPD band. RAPDs predominantly provide dominant markers but homologous allele combinations can sometimes be iden-tified with the help of detailed pedigree information.

RAPDs have several advantages and for this reason they are widely used (Karp and Edwards, 1997). (i) Neither DNA probes nor sequence information is required for the design of specific primers. (ii) The proce-dure does not involve blotting or hybridiza-tion steps thus making the technique quick, simple and efficient. (iii) RAPDs require rel-atively small amounts of DNA (about 10 ng per reaction) and the procedure can be auto-mated; they are also capable of detecting higher levels of polymorphism than RFLPs.

(iv) Development of markers is not required and the technology can be applied to vir-tually any organism with minimal initial development. (v) The primers can be uni-versal and one set of primers can be used for any species. In addition, RAPD products of interest can be cloned, sequenced and con-verted into other types of PCR-based mark-ers such as sequence tagged sites (STS), sequenced characterized amplified regions (SCAR), etc.

Reproducibility affects the way in which RAPD bands can be standardized for compar-ison across laboratories, samples and trials and whether RAPD marker information can be accumulated or shared. Due to frequently observed problems with reproducibility of overall RAPD profiles and specific bands, this marker class is often treated with reserve. In replication studies by Pérez et al.

(1998), mispriming error amounted to 60%.

Several factors have been shown to affect the number, size and intensity of bands.

These include PCR buffers, deoxynucleo-tide triphosphates (dNTPs), Mg²⁺ concen-tration, cycling parameters, source of Taq polymerase, condition and concentration of template DNA and primer concentra-tion. Results obtained by RAPDs are highly prone to user error and bands obtained can vary considerably between different runs of the same sample. To correct the problems that may be encountered when carrying out RAPD-PCR, it is important to bear in mind the following: (i) the concentration of DNA can alter the number of bands; (ii) RAPD profiles vary depending on the Mg²⁺ con-centration and the PCR buffer provided by Taq polymerase suppliers may or may not contain Mg²⁺ ions; (iii) there are different sources of Taq polymerase and there is great variation between profiles produced using Taq polymerase obtained from different companies; (iv) there are a large number of alternative cycling times and temperatures which are equally important and depend on the type of machine used and even the wall thickness of the PCR tubes.

Generally if a PCR does not work there is likely to be something wrong with the template DNA, primers, Taq polymerase or choice of conditions. Initially it is impor-tant to try and repeat the PCR under the same conditions to ensure that there was not a simple error that resulted in the fail-ure. In addition it is recommended that both positive and negative controls are included.

A positive control with a template known to amplify well will ensure that all reagents have been added and that they are all func-tioning. A negative control without template DNA will reveal any contamination. In most cases if the PCR does not work and it is not

clear what might be causing the problem, it is worth starting from the beginning by dis-posing of all the reagents used and preparing fresh ones. A careful experiment revealed that reproducibility could be improved and Taberner et al. (1997) reported that 3396 out of 3422 bands (99.2%) were reproducible.

On the other hand, low reproducibility is a major limitation of RAPD markers, par-ticularly in ongoing genetic and plant breed-ing programmes in which the accumulated information and markers and marker data are shared between laboratories and experi-ments. RAPD markers may still find their applications in independent genetic diver-sity and phylogenetic studies that do not depend on data sharing or accumulation. As RAPD markers can be converted into other types of markers, they have a unique role in the development of target markers for crop species that have limited molecular markers available to cover the whole genome.

To overcome the problem associated RAPD analysis, Paran and Michelmore (1993) converted RAPD fragments into simple and robust PCR markers known as SCARs. This procedure increases the repro-ducibility of RAPD markers and also avoids the occurrence of non-homologous mark-ers of equal molecular weight. These spe-cific markers are obtained by introducing RAPD bands (polymorphic) into single markers which are then sequenced and specific primers are designed usually by expanding the original decamer primer sequence with 10–15 bases so that only the band of interest is amplified. In general, DNA can be isolated from agarose gels, cloned and sequenced to produce the start-ing DNA template for the development of a variety of PCR-based markers. The cloned and sequenced DNA fragments can then be used for the development of CAPS, single strand conformation polymorphism (SSCP) or SNP markers.

AFLP

Amplified fragment length polymorphism (AFLP; Zabeau and Voss, 1993; Vos et al., 1995) is based on the selective PCR ampli-fication of restriction fragments from a total

double-digest of genomic DNA under high stringency conditions, that is, the combi-nation of polymorphism at restriction sites and hybridization of arbitrary primers, and because of this AFLP is also called selective restriction fragment amplification (SRFA).

It was perfected by the company Keygene in the Netherlands for initial use in plant improvement and has been patented. The AFLP technique combines the power of RFLP with the flexibility of PCR-based markers and provides a universal, multi-locus marker technique that can be applied to complex genomes from any source. The method is based on the identification of AFLP using selective PCR amplification of digested/ligated genomic or cDNA templates separated on a polyacrylamide gel, includ-ing restriction–ligation, pre-amplification and selective amplification (Fig. 2.3). The purified genomic DNA is first cleaved with one or more restriction endonucleases, i.e. a 6-cutter (EcoRI, PstI and HindIII) and a 4-cutter (MseI, TaqI). Adaptors of 18–20 bp and of known sequence, adapted at the sticky ends of the restriction sites, are then added to the ends of DNA fragments by a ligation reaction using T4 DNA ligase. DNA amplification is carried out using primers with the sequence specificity of the adaptor to generate a subset of fragments of differ-ent sizes (∼up to 1 kb). The primer(s) also contains one or more bases at their 3' ends that provide amplification selectivity by limiting the number of perfect sequence matches between the primer and the pool of available adaptor/DNA templates. The resulting amplification products (50–400 bp size range) are typically observed by radio-labelling one of the primers followed by fragment separation on acrylamide gels to identify polymorphisms (changes in restric-tion sizes).

An AFLP primer is composed of a synthetic adaptor sequence, the restric-tion endonuclease recognirestric-tion sequence and an arbitrary, non-degenerate ‘selec-tive’ sequence (typically one, two or three nucleotides). In the first step, 500 ng of genomic DNA will be completely digested with two restriction enzymes, one fre-quent cutter (4-bp recognition site) and

GAATTC

+ EcoRI and MseI

TTA

Pre-amplification EcoRI Primer 1

MseI Primer 1

Fig. 2.3. AFLP flowchart. Adaptor DNA = short double strand DNA molecules, 18–20 bp in length, representing a mixture of two types of molecules.

Each type is comparable with one restriction enzyme generated DNA end. Pre-amplifications uses selective primers, which contain an adaptor DNA sequence plus one or two random bases at the 3' end for reading into the genomic fragments.

Primers for re-amplification have the pre- amplification primer sequence plus one or two additional bases at the 3' end. A tag (*) is attached at the 5' end of one of the re-amplification primers for detecting amplified molecules.

one rare cutter (6-bp recognition site).

Oligonucleotide adaptors are ligated to the end of each restriction DNA which serve together with restriction site sequences as target sites for primer annealing, one end

with a complementary sequence for the rare cutter and the other with the complemen-tary sequence for the frequent cutter. In this way only fragments which have been cut by the frequent cutter and rare cutter will be amplified. Primers are designed from the known sequence of the adaptor, plus one to three selective nucleotides which extend into the fragment sequence. Sequences not matching these selective nucleotides in the primer will not be amplified so that the specific amplification of only those frag-ments matching the primers is achieved.

The option to permutate the order of the selective bases and to recombine the prim-ers with each other will theoretically lead to the gradual collection of all restriction fragments from a particular enzyme com-bination that is of a suitable size for DNA fragment analysis from a genotype. The multiplex ratio of an AFLP assay is a func-tion of the number selective nucleotides in the AFLP primer combination, the selective nucleotide motif, GC content and physical genome size and complexity. Typically, two selective nucleotides are used for species with small genomes (1 × 10⁸–5 × 10⁸ bp), e.g. Arabidopsis thaliana L. (1 × 10⁸ bp) and rice (Oryza sativa L.) (4 × 10⁸ bp), and three selective nucleotides are used for species with large genomes (5 × 10⁸–6 × 10⁹ bp), e.g. maize, soybean, sunflower and many others. It is theoretically possible to use several tens of combinations of restriction enzymes at sites of four to six bases and a large number of combinations of selective bases on the amplification primers. Thus, as indicated by Falque and Santoni (2007), the restriction–amplification combinations are nearly infinite.

AFLP products can be separated in high-resolution electrophoresis systems. The number of bands produced can be manipu-lated by the number of selective nucleotides and the nucleotide motifs used. A well-balanced number of amplified restriction fragments ranges from 50 to150 bp. A major improvement has been made by switching from radioactive to fluorescent dye-labelled primers for the detection of fragments in gel-based or capillary DNA sequencers in which fluorescently labelled fragments pass

the detector near the bottom of the gel/end of the capillary, resulting in a linear spac-ing of DNA fragments and therefore increas-ing the resolution over the whole size range (Schwarz et al., 2000).

In general, AFLP assays can be carried out using relatively small DNA samples (typically 1–100 ng per individual). AFLP has a very high multiplex ratio and genotyp-ing throughput and is relatively reproduc-ible across laboratories. Simple off-the-shelf technology can be applied to virtually any organism with no formal marker devel-opment required and in addition, a set of primers can be used for different species.

However, there are limitations to the AFLP assay. (i) The maximum polymorphic infor-mation content for any bi-allelic marker is 0.5. (ii) High quality DNA is needed to ensure complete restriction enzyme diges-tion. Rapid methods for isolating DNA may not produce sufficiently clean template DNA for AFLP analysis. (iii) Proprietary technology is needed to score heterozygotes and++ homozygotes, otherwise AFLPs must be dominantly scored. (iv) AFLP markers often cluster densely in centromeric regions in species with large genomes, e.g. barley (Qi et al., 1998) and sunflower (Gedil et al., 2001). (v) Developing locus-specific mark-ers from individual fragments can be dif-ficult. (vi) AFLP primer screening is often necessary to identify optimal primer spe-cificities and combinations otherwise the assays can be carried out using off-the-shelf technology. (vii) There are relatively high technical demands in AFLP analysis includ-ing radio-labellinclud-ing and skilled manpower.

(viii) Marker development is complicated and not cost-effective. (ix) Reproducibility is relatively low compared to RFLP and simple sequence repeat (SSR) markers but better than RAPD marker as AFLP reveals large numbers of bands and not all the bands will be comparable across laboratories or trials due to potential false positive, false negative and complicated gel backgrounds.

The AFLP technique can be modified so that one primer is obtained from a known multi-copy sequence to detect sequence-specific amplification polymorphisms. This approach was used successfully to generate

genome-wide Bare-1 retrotransposon-like markers in barley (Waugh et al., 1997) and diploid Avena (Yu and Wise, 2000) as well as in lucerne by making use of consen-sus sequences from long terminal repeats (LTRs) of Tms1 retrotransposon (Porceddu et al., 2002). The cDNA-AFLP technique (Bachem et al., 1996) which applies the standard AFLP protocol to a cDNA tem-plate, was used to display transcripts whose expression was rapidly altered during race-specific resistance reactions, for the isola-tion of differentially expressed genes from a specific chromosome region using aneu-ploids and for the construction of genome-wide transcription maps (as reviewed by Mohler and Schwarz, 2005). In addition, there are several modified AFLP tech-niques based on the use of endonucleases such as single endonuclease (MspI) AFLP (Boumedine and Rodolakis, 1998), three endonuclease-AFLP (van der Wurff et al., 2000), and second digestion AFLP (Knox and Ellis, 2001). Developments in the detection of AFLP include the re placement of radio-active detection with silver stain-ing, fluorescent AFLP or agrarose gels for single endonuclease AFLP. Recent studies have addressed specific areas of the AFLP technique including comparison with other genotyping methods, assessment of errors, homoplasy, phylogenetic signal and appro-priate analysis techniques. The study by Meudt and Clarke (2007) provides a syn-thesis of these areas and explores new directions for the AFLP technique in the genomic era.

SSR

Microsatellites, also known as SSRs, short tandem repeats (STRs) or sequence-tagged microsatellite sites (STMS), are tandemly repeated units of short nucleotide motifs that are 1–6 bp long. Di-, tri- and tetranu-cleotide repeats such as (CA)_n, (AAT)_n and (GATA)_n are widely distributed through-out the genomes of plants and animals (Tautz and Renz, 1984). One of the most important attributes of microsatellite loci is their high level of allelic variation, mak-ing them valuable as genetic markers.

The unique sequences bordering the SSR motifs provide templates for specific prim-ers to amplify the SSR alleles via PCR.

Referred to as simple sequence length poly-morphisms (SSLPs), they pertain to the number of repeat units that constitute the microsatellite sequence. The rates of muta-tion of SSR are about 4 × 10⁴–5 × 10⁶ per allele and per generation (Primmer et al., 1996). The predominant mutation mecha-nism in microsatellite tracts is ‘slipped-strand mispairing’ (Levinson and Gutman, 1987). When slipped-strand mispairing occurs within a microsatellite array during DNA synthesis, it can result in the gain or loss of one or more repeat units depending

Referred to as simple sequence length poly-morphisms (SSLPs), they pertain to the number of repeat units that constitute the microsatellite sequence. The rates of muta-tion of SSR are about 4 × 10⁴–5 × 10⁶ per allele and per generation (Primmer et al., 1996). The predominant mutation mecha-nism in microsatellite tracts is ‘slipped-strand mispairing’ (Levinson and Gutman, 1987). When slipped-strand mispairing occurs within a microsatellite array during DNA synthesis, it can result in the gain or loss of one or more repeat units depending