Oligonucleotide probes allele-specific

Saiki, R. K., Chang, C., Levenson, C. H., Warren, T. C., Boehm, C. D. etal.. Diagnosis of sickle cell anemia and (3-thalassemia with enzymatically amplified DNA and non radioactive allele-specific oligonucleotide probes. N. Engl. J. Med. 319, 537-541 (1988). 

Figure 1-7-8. Comparison of Probes Single-Gene Versus Allele-Specific Oligonucleotide (ASO)...

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... 

For additional discussion of allele-specific oligonucleotide (ASO) probes and dot blots, see Section I, Chapter 7 Genetic Testing. 

Saiki, R., Bugawan, T.L., Horn, G.T., Mullis, K.B., Ehrlich, H.A. (1986) The analysis of enzymatically labeled / -globin DQa DNA with allele-specific oligonucleotide probes. Nature 324, 163-166. 

D. Dot blot using allele specific oligonucleotide probes... 

M.S. Ristaldi, M. Pirastu, C. Rosatelli, G. Monni, H. Erlich, R. Saiki, A. Cao, Prenatal Diagnosis of Beta-thalassaemia in Mediterranean Populations by Dot Blot Analysis with DNA Amplification and Allele Specific Oligonucleotide Probes , Prenat. Diagn, 9(9), 629-638 (1989). 

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. 

Saiki RK, Bugawan TL, Horn GT, Mullis KB, Erlich HA. Analysis of enzymatically amplified beta-globin and HLA-DQ alpha DNA with allele-specific oligonucleotide probes. Nature 1986 324 163-166. 

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.

Step 3. Design the 25 oligonucleotides shown in Table 4, including four common probes, three extra probes, and 18 allele-specific probes. Divide all these probes into three groups (groups 16, 26, and 31) (Table 4). 

Weisgraber KH, Newhouse YM, Mahley RW. Apolipoprotein E genotyping using the polymerase chain reaction and allele-specific oligonucleotide probes. Biochem Biophys Res Commun 1988 157 1212-17. 

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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