Allele-specific hybridization

Prince JA, Feuk L, Howell WM, Jobs M, Emahazion T, Blennow K, Brookes AJ. Robust and accurate single nucleotide polymorphism genotyping by dynamic allele-specific hybridization (DASH) design criteria and assay validation. Genome Res 2001 11 152-162. 

Jobs M, Howell WM, Stromqvist L, Mayr T, Brookes AJ. DASH-2 flexible, low-cost, and high-throughput SNP genotyping by dynamic allele-specific hybridization on membrane arrays. Genome Res 2003 13 916-924. 

Dynamic allele-specific hybridization, a method to detect SNPs, is based on dynamic heating and coincident monitoring of DNA denaturation and avoids the use of additional enzymes or reaction steps. 

W. Matthias Howell, Magnus Jobs, Ulf Gyllensten and Anthony J. Brooks, Dynamic allele-specific hybridization, Nature Biotechnology, 17 (1999), 87-88. 

Essentially all genotyping approaches used to date fall into one of four categories allele-specific hybridization, primer extension, oligonucleotide ligation, or invasive cleavage. Figure 5.7-7 illustrates these different approaches. We will first... 

Allele-specific Hybridization. Allele-specific hybridization distinguishes the allele present at an SNP using two different oligonucleotide probes. Each probe... 

DASH, dynamic allele-specific hybridization AS-PCR, allele-specific polymerase chain reaction AS-PE, allele-specific primer extension APEX, arrayed primer extension FP-TDI, fluorescence polarization template directed dye terminator incorporation MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry OLA, oligonucleotide ligation assay RCA, ... 

Flowell WM, etal. Dynamic allele-specific hybridization. A new method for scoring single-nucleotide polymorphisms. Nat Biotechnol 1999 17 87-88. 

Earlier reports on the use of MALDI-TOF-MS for the detection of mutahons and/or sequence changes have employed more conventional techniques such as restriction endonuclease digests [146] or the ligase chain reaction [147]. Inihally, allele-specific hybridization was also combined with mass spectrometric detection. Smith and coworkers, for example, used pephde NA probes hybridized against an immobilized PCR template for genotyping [148]. Finally, the direct measurement of PCR products has been attempted as a means of NA analysis and genotyping [17, 24, 92, 149, 150]. 

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... 

Fig. 4. Illustration of the multiplex allele specific diagnostic assay. At the top of the panel, radioactive ohgonucleotide probes (indicated by stars) are selected by hybridization to amphcons from patient samples affixed to membrane filters. When a putative mutation-bearing allele hybridizes to a radioactive probe, it can be eluted from the filter and subjected to sequencing using chemical or radioactive dideoxy-terminator methods. This permits unequivocal identification of a large number of mutations at a high throughput.

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.

As an additional characterization, we hybridize the allele-specific probes to rDNA Southern blots to confirm that they hybridize to restriction fragments containing the region from which they were designed. The Southern blots are hybridized and washed using the same conditions described for RNA slot blots. 

---