Microarray Analysis

Distinguishing alleles by hybridization takes advantage of the different thermal stabilities of a hybrid between an allele-specific probe and the SNP-containing sequence (Fig. 3). One approach to carry out allele-specific hybridization on microarrays is the GeneChip® assay introduced by Affymetrix.

On high-density microarrays thousands of allele-specific oligonucleotides complementary to a target sequence are attached to a solid surface. For each allele of a SNP, at least four oligomers are designed, differing only in the interrogating position. Additionally, in many cases a series of oligonucleotides walking over each variant of the SNP are used. The target sequence is amplified incorporating fluorescence-labeled nucleotides and hybridized to the array. Subsequently, the array is scanned to measure the fluorescence intensity for each hybrid. The reference sequence is expected to hybridize more efficiently to the corresponding probe and, therefore, gives stronger fluorescent signals than single base mismatches. These mismatch probes serve as control for cross-hybridization. The presence of homozygotes and heterozygotes give rise to different hybridization patterns with the probe complementary to the reference sequence showing the highest fluorescence intensity. In the presence of a sample with a substituted variant, the probe containing the complementary variant base will obtain the highest fluorescence intensity.

Hybridization-based genotyping can be an efficient method to monitor a large number of SNPs. The sophisticated technology of photolithography and solid-phase DNA synthesis allows construction of arrays carrying up to 260,000 features on a 1.28 x 1.28 cm array. Therefore, it seems to be feasible to rapidly screen thousands of SNPs using microarrays. The GeneChip HuSNP™ offers interrogation of nearly 1500 SNPs throughout the human genome. However, a drawback of the array format is the low flexibility in establishing an assay for a SNP. Due to high set-up costs of a project, it is difficult to add new SNPs or replace existing assays.

Another limitation is that the assays may fail to distinguish between homozygous and heterozygous, and deletions and insertions will not be detected as with pyrosequen-cing. A future challenge is to improve accuracy and decrease the false positive rate. The sensitivity of microarray-based hybridization is dramatically influenced by the target sequence, including inter- and intramolecular structures and also the presence of repetitive sequence elements. To allow more robust genotyping results dynamic allele-specific hybridization (DASH) monitoring duplex formation over a temperature gradient has been developed (1,2). Despite some disadvantages, future applications of microarray genotyping will be well established and custom-tailored medium density arrays for routine diagnostic screenings or pharmacogenetic studies.

Cure Your Yeast Infection For Good

Cure Your Yeast Infection For Good

The term vaginitis is one that is applied to any inflammation or infection of the vagina, and there are many different conditions that are categorized together under this ‘broad’ heading, including bacterial vaginosis, trichomoniasis and non-infectious vaginitis.

Get My Free Ebook

Post a comment