Allele specific oligonucleotide assays

DNA Isolation Techniques for Allele-Specific Oligonucleotide Assay or Gel Denaturing and Polymerase Chain Reaction... 

Techniques used to analyze DNA, RNA, and proteins. ASO = allele-specific oligonucleotides. ELISA = enzyme-linked immunosorbent assays. 

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. 

Green ETC, Bain SC, Day PJ, Barnett AH, Charleson F, Jones AF, Walker MR. Detection of human apolipoprotein E3, E2, and E4 genotypes by an allele-specific oligonucleotide-primed polymerase chain reaction assay development and validation. Clin Chem 1991 37 1263-8. 

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. 

Oligonucleotide Ligation Assay Allele-specific Common /... 

Figure 40-10 Methylation-specific PCR assay for the diagnosis of PWS and AS, Extracted DNA is treated with sodium bisulfate before amplification using multiplex PCR and oligonucleotide primers specific for modified DNA. Normal individuals show amplicons representing their methylated maternal allele and amplicons from their unmethylated paternal allele. PWS patients show only the maternal allele and AS patients show only the paternal allele. Results observed following PCR amplification and gel electrophoresis of patients referred for PWS and AS testing. Patient DNA with patterns diagnostic of AS (/ones / and 5),...

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... 

In the assay, three oligonucleotides are designed for each SNP locus. Two oligos are specific to each allele of the SNP site, called the Allele-Specific Oligos (ASOs). 

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, ... 

Allele-specific expression levels can be assessed in two ways. Expression of mRNA and cDNA with different alleles at heterozygote SNPs is assessed using oligonucleotides and TaqMan/real-time PCR 5 nuclease assays (ABI, Foster City, CA) or the primer extension method (SnapShot, ABI, Foster City, CA and MALDI TOF, Sequenome). 

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