PCR based molecular markers

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Random amplified polymorphic DNA (RAPDs) are in fact just one example of a whole set of PCR-based molecular markers, which have been collectively termed as MAAP, multiple arbitrary amplicon profiling (Caetano-Anolles, 1993). These approaches have in common the use of one, usually, or two primers of random sequence to amplify multibanded fingerprints from a complex genome (Fig. 1.8). The techniques differ in the length and sequence of the primers, number of amplification cycles, temperature of the annealing stage and methods for evidencing polymorphisms (Caetano-Anolles, 1993, 1994). The 'classic' RAPD technique (Williams et al., 1990) uses one single primer, 10-nt long, with a GC percentage between 50 and 70%, to amplify in a low-stringency reaction (annealing at 34-360C) sequences encompassed by imperfect inverted repeats of the primer. This technique has been applied to all types of organisms for different purposes. Its main advantages are that it is fast, easy to perform and

Glass Main Door PlanSingle Bands Rapd
Fig. 1.8 Possible outcomes of PCR amplification from a single randomly chosen oligonucleotide in RAPD or AP-PCR technique. Results of gel electrophoresis evidence appearance or disappearance of bands in the profile according to different types of mutation in the template DNA.

requiring small amounts of DNA. The disadvantages are the dominance of RAPD markers and the poor reproducibility of RAPD patterns in different laboratories. The AP-PCR technique (Welsh and McClelland, 1990) uses a longer primer of arbitrary sequence in a reaction that includes some cycles at a lower annealing temperature. A particular modification could involve the use of two different random primers, thereby increasing the number of bands in the fingerprint (Micheli et al., 1993; Hu et al., 1995; Diaz and Ferrer, 2003).


Sequence characterised amplified regions (SCARs) were first described in 1993 (Paran and Michelmore, 1993), shortly after the invention of RAPD markers. A



Pcr Based Molecular Marker
Digestion with cn/vmc R


Fig. 1.9 Application of SCAR markers, PCR amplification followed by restriction with endonucleases evidences the homozygotic (left and centre) or heterozygotic (right) state of the individual.

SCAR is identified by two specific primers that amplify a well-defined genetic locus, derived by sequencing a RAPD product. This type of technique (Fig. 1.9) exploits first the advantages of RAPDs in terms of simplicity and rapidity. When the interesting RAPD markers have been singled out, they are sequenced, also partially, and the sequence information used to design long primers, 24-25 nt, to amplify that locus in a specific way. The polymorphism between SCAR bands is determined by variation in length of the sequence between the two primers, or by lack of annealing. In some cases therefore SCARs can be co-dominant. However, SCARs amplified by different individuals can also be of the same length, because the longer primers will anneal even in presence of few mismatches, as opposed to the short primers used in RAPDs. In this case, polymorphisms in the intervening sequence can be evidenced by restriction with enzymes with four-bp recognition sequences (PCR-RFLP). Not all RAPDs markers are amenable to transformation into SCAR markers. RAPD polymorphisms can also arise with different mechanisms, for example rearrangement of secondary structures during amplification rounds (Caetano-Anoll├ęs, 1993).

SSR (simple sequence repeats) are also called microsatellites, and consist of 110 bp units repeated in tandem, with a variable number of repetitions (Tautz, 1989). Usually they represent a single locus, which is hypervariable and highly polymorphic in different individuals. They can be detected by amplification from specific primers annealing to the unique flanking sequences and this can make them amenable to transfer to related species. Their discovery implies the construction of genomic libraries that are then selected with oligonucleotides containing the repetitive motif and enriched in several ways. Sequencing of the positive clones yields information on the length and on the flanking sequences, which have to be unique in the genome. Since microsatellites variability derives from 'slippage' during the replication process, the same problem can also occur during the PCR reaction, producing a profile of 'stuttering' bands, differing in length of 1-5 repetitions. Detection of SSRs is accomplished with a sequencing apparatus, either on gel or in capillaries. SSRs are an example of locus-specific PCR, but they have been also transformed into a 'random' approach by employing a microsatellite sequence as primers, adding 3-4 selective bases (Inter-SSR PCR, Zietkiewicz et al., 1994).


AFLPs, amplified fragment length polymorphisms (Vos et al., 1995), are based on the detection by PCR amplification of restriction fragments generated from a genome. The amplification of the restriction fragments is obtained by ligating specific adapters to the fragments' ends and utilising primers complementary to the adapters. To decrease the number of bands amplifiable from a complex genome, the primers include at the 3' end 1-3 selective bases extending into the restriction fragments past the adapter. Therefore, a subsample of restriction fragments is 'selected' during amplification. By separating the amplification products on long polyacrylamide gels, 50-100 different bands can easily be detected and scored. The origin of polymorphism in AFLP is the same as in RFLP: changes in the restriction site sequence or insertions/deletions between two adjacent restriction sites. Additionally, polymorphisms in the adjacent bases will prevent amplification, according to the selective bases of the primers. AFLPs are also dominant markers, and they are more laborious to produce: DNA has to be of high quality and high molecular weight, the production involves several steps, and detection requires technical ability in casting the gel and staining it.

SNP, single nucleotide polymorphism, are single base changes in the DNA sequences, the most abundant type of mutation. In general, they represent biallelic systems, with two allelic forms in each locus, but systems with higher numbers of alleles have been described (Wang et al, 1998). The informational content is not very high, but it can be increased by the occurrence of several adjacent SNPs in one sequence. They can be identified by sequencing, and then a procedure for identifying them with PCR is designed. For instance, if the 3'-most base of one primer anneals at the SNP site, different oligonucleotides can discriminate between alleles, since only the matching primer will be able to amplify the sequence. The search for SNP can be more effectively carried out by hybridisation on chips in macro- or microarrays, using oligonucleotide probes

(Lemieux et al., 1998). The advantage of SNPs is mostly in their high number and density along the genome, giving the possibility of developing haplotyping systems for genes of interest.

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