Allele-specific oligonucleotide hybridization

Hruban RH, van Mansfeld AD, Offerhaus GJ, et al. K-ras oncogene activation in adenocarcinoma of the human pancreas. A study of 82 carcinomas using a combination of mutant-enriched polymerase chain reaction analysis and allele-specific oligonucleotide hybridization. Am J Pathol. 1993 143 545-554. 

RT-PCR, reverse transcription-polymerase chain reaction ASO, allele-specific oligonucleotide hybridization SSCP, single-stranded conformational polymorphism analysis. 

The basis of the allele-specific oligonucleotide (ASO) assay is that DNA duplexes which contain a mismatch are destabilized and have a lower melting temperature than correctly paired duplexes. To test for mutations using ASO, two probes, one containing the normal sequence and one containing the mutant sequence, are produced and hybridized to the patient s DNA. For each normal and mutant probe, conditions can be found where the probe will hybridize to only its perfectly matched duplex. If the patient sample contains only normal sequence, only the normal probe will hybridize. In a heterozygous sample, both the mutant and normal probes will hybridize, and in a homozygous mutant sample only the mutant probe will hybridize. 

Fig. 2. Analysis of parental and hybrid maize rRNAs with allele-specific oligonucleotide probes. In the cross illustrated the maternal parent P-1 is B73 and the paternal parent P-2 is Mo 17. The samples were applied at 0.5 /ig/slot with six replicates of each. The parental and hybrid samples are identified. (A) the oligonucleotide probe used is the same one shown in Fig. 1A. In (B) the probe is the same as in Fig. IB.

With one common mutation, a very successful and efficient hybridization analysis is the PCR allele-specific oligonucleotide (ASO) assay. This requires that the sequence of the common mutation is known. 

This technique, described in Section I, involves the construction of short DNA sequences (oligonucleotides) of approximately 20 bp that exactly complement either the mutated sequence or the normal sequence (hence, allele-specific ). The labeled oligonucleotide is then used to probe DNA from the individual in question. This DNA may be placed, for example, on a dot blot. Successful hybridization of the ASO containing the mutation indicates presence of the mutation, whereas hybridization of the normal ASO indicates presence of the normal sequence. Hybridization of both probes would be seen in a heterozygote. Because a different probe must be constructed for each mutation, this technique is practical when a limited number of mutations... 

To circumvent the disadvantages of SOLAC-LOS, SOLAC-GOS was developed. In this scheme, all allele-specific short oligonucleotides were immobilized on chips, hybridization and ligation were incorporated into a single step, and the gain of signal signifies the presence of mutations (Fig. 3). 

The specific way that SNPs can be identified varies between different brands. The most common ones are allele discrimination by hybridization in Affymetrix arrays (56) and allele-specific extension and ligation to a bar-code oligonucleotide hybridized to a universal array, as is done by the Illumina GoldenGate BeadArray Assay (57). 

OLA. The OLA uses an enzymatic reaction to increase the specificity of a hybridization-based approach. Three very specific oligonucleotide probes are used in OLA one specific for the wild-type allele, one specific for the variant allele, and a common probe that carries a fluorescent label. PCR is used to create amplicons containing the polymorphic site. When the PCR products are incubated with all three probes, the 5 region of the common probe anneals just downstream of the polymorphic site. The 3 end of either of the allele specific probes anneals adjacent to the 5 end of the common probe. In the presence of thermostable DNA ligase, the two probes will join only if there is a perfect match. The results of the assay can be observed either by gel... 

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