Genotyping On Electronic Microarrays

Detection of single-nucleotide polymorphisms (SNPs) on electronic microarrays is achieved via hybridization of allele-specific probes to biotinylated, amplified target DNA. Genomic DNA is extracted from the sample, and

Biotinylated DNA target

• Permeation Layer with Streptavidin

Platinum electrode

Fig. 1 (A) The NanoChip cartridge, NanoChip electronic microarray, and schematic cross section of an individual test site (not to scale). The NanoChip cartridge contains the microarray, fluidic channels and ports for automated addition of samples and reagents, and electronic interfaces. As depicted in the drawing of the test site, a permeation layer containing streptavidin covers the surface of the microarray, which enables attachment of biotinylated DNA targets. (B) The NanoChip molecular biology workstation. (View this art in color at www.dekker.com.)

Biotinylated DNA target

• Permeation Layer with Streptavidin

Platinum electrode

Fig. 1 (A) The NanoChip cartridge, NanoChip electronic microarray, and schematic cross section of an individual test site (not to scale). The NanoChip cartridge contains the microarray, fluidic channels and ports for automated addition of samples and reagents, and electronic interfaces. As depicted in the drawing of the test site, a permeation layer containing streptavidin covers the surface of the microarray, which enables attachment of biotinylated DNA targets. (B) The NanoChip molecular biology workstation. (View this art in color at www.dekker.com.)

the target sequence containing the region of interest is amplified by PCR. The use of one biotinylated amplification primer and one unmodified amplification primer generates a product that is biotinylated on one strand. A wide range of target size (from <100 base pairs to >1000 base pairs) can be accommodated on electronic micro-arrays. For genotyping analysis of two or more targets from a sample, multiple oligonucleotide primer pairs can be used in a single amplification reaction, which will generate multiple biotinylated DNA targets.

After the amplification reaction, desalting with size exclusion membranes is performed to facilitate electronic transport and remove unincorporated amplification primers. The biotinylated targets are then electronically addressed onto NanoChip cartridges. Software on the workstation enables the user to designate the test sites to be addressed for each sample. Electronic activation concentrates the biotinylated DNA in the sample to the test sites, where the DNA remains anchored via interaction with the streptavidin in the permeation layer. Unbound nucleic acids are removed by a series of fluidic washes before addressing the next sample. When multiple NanoChip cartridges are processed in a single loader run, cartridges are loaded in parallel, minimizing the time required for electronic addressing.

One method for sample genotyping uses fluorescently labeled reporter probes specific for the wild-type and variant sequences (Fig. 2). For target sequences containing significant secondary structure near the SNP, an unmodified ''stabilizer'' oligonucleotide can be used; this oligonucleotide hybridizes to the target sequence immediately adjacent to the nucleotide of interest (Fig. 2). Hybridization of the stabilizer next to a perfectly matched reporter probe generates a base-stacking energy component that, added to the Watson-Crick base-pairing energies, further stabilizes the reporter/target interaction. An alternative method utilizes chimeric ''discriminator'' oligonucleotides that contain a target-specific region, complementary to the wild-type or variant alleles, and one of two universal tail sequences. Two fluorescently labeled universal reporter probes, each complementary to one of the tail sequences, are used to detect the bound discriminators.

Whether allele-specific reporter probes or allele-specific discriminator oligonucleotides/universal reporter probes are utilized, genotype determination is achieved by monitoring the fluorescent signal remaining on each test site after the mismatched allele-specific oligonucleo-tides have been destabilized. Mismatched probes are removed by increasing the stringency of the environment, either thermally or electronically; perfectly matched probes will remain hybridized under the more stringent conditions. The NanoChip reader uses a two-laser system to detect the fluorescent signal remaining at each test site.

Fig. 2 Genotyping on electronic microarrays. The region of interest is amplified from genomic DNA by using one biotinylated amplification primer. After electronic addressing and denaturation, allele-specific reporter probes or allele-specific discriminator oligonucleotides/universal reporter probes are used for genotype determination (see text for detail). The use of stabilizer oligonucleotides is optional.

Fig. 2 Genotyping on electronic microarrays. The region of interest is amplified from genomic DNA by using one biotinylated amplification primer. After electronic addressing and denaturation, allele-specific reporter probes or allele-specific discriminator oligonucleotides/universal reporter probes are used for genotype determination (see text for detail). The use of stabilizer oligonucleotides is optional.

Predominance of fluorescent signal from one labeled reporter indicates a sample that is homozygous for the corresponding allele, whereas equivalent levels of fluorescent signal from both reporter oligonucleotides indicate a heterozygous genotype.

For multiplex genotyping, locus-specific discriminator (or reporter) pairs can be hybridized and discriminated sequentially (S.A. Williams, Children's Hospital of Orange County, personal communication).1-11-1 Such analyses can be performed on a single DNA target containing multiple SNPs or on different DNA targets amplified from a single sample. As many as seven genotyping assays have been performed on a single 1.2-kb target (Dr. Dennis O'Kane, Mayo Clinic, personal communication). The ability to perform multiplex genotyping analyses greatly increases the capacity of the 100-site array, such that hundreds of genotypes can be generated without having to perform separate amplification and addressing reac-

tions.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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