Assay Formats Direct Hybridization

Several formats of wet-ware were adopted to bead-based nucleic acid testing. The simplest method is the direct hybridization of labeled nucleic acids to beads carrying a sequence-specific oligonucleotide.

An assay for gene expression analysis using color-coded microspheres was developed using this technique. In Ref. [4], labeled cRNA was generated which was hybridized to a set of 20 bead populations carrying specific capture sequences of 25 bp for 20 Arabidopsis genes. The obtained expression profiles were similar to those obtained by Affymetrix GeneChip Analysis.[4]

In addition to expression profiling, sequence-specific hybridization of labeled PCR product to microspheres has been performed. A sensitive multiplexed bead assay for the detection of three viral nucleic acids (HIV, HSV, and HCV) was developed. Here labeled primers were used in the amplification of the relevant viral nucleic acids, which were hybridized to sequence-specific oligonucleotides bound to the beads.[5]

A third test applying the format of direct hybridization was a screening test for mutations in the CFTR gene,

The simultaneous and efficient hybridization of oligonu-cleotides with different lengths and GC contents is still a problem, even when using special hybridization buffers. In addition, the allelic discrimination of single nucleotide polymorphisms by hybridization is problematic because of the similarity of the sequences. To this end, the idea of using so-called zip codes was brought to light. Here, probes for allelic discrimination carry a 5'-dangling end with an artificial unique DNA sequence for hybridization to beads subsequent to allelic discrimination. These zip codes are designed with identical length and GC content and limited cross-talk in simultaneous hybridization to their complementary sequence bound to the bead. An additional advantage is that the development of novel assays no longer requires production of differently coated beads. With this assay format, three different techniques have been evaluated: the oligo ligation assay (OLA), minisequencing (also called single-base chain extension, SBCE), and the allele-specific primer extension (ASPE).

In the OLA, two adjacent hybridized oligonucleotides are enzymatically ligated to each other using DNA ligase. This reaction only takes place when the nucleotides next to the ligation position are fully complementary. The first (capture) oligonucleotide is designed to hybridize to the target amplicon with its 3' base complementary to the polymorphic base. It also carries a 5'-dangling end with the zip code. The second (reporter) oligonucleotide

Fig. 2 Principle of the ASPE reaction. A) For each allele, one primer is designed carrying a 5'-dangling end with a unique zip code sequence. B) Only a primer hybridized with a perfect match at the 3' end will be elongated and labels will be incorporated. C) Subsequent to allelic discrimination, primers are captured by color-coded beads carrying complementary zip code sequences.

Fig. 2 Principle of the ASPE reaction. A) For each allele, one primer is designed carrying a 5'-dangling end with a unique zip code sequence. B) Only a primer hybridized with a perfect match at the 3' end will be elongated and labels will be incorporated. C) Subsequent to allelic discrimination, primers are captured by color-coded beads carrying complementary zip code sequences.

hybridizes to the target sequence just adjacent to the 3' end of the capture probe and carries a fluorescent tag at its 3' end. If the capture probe matches exactly the SNP query position, the probes are ligated to each other, bringing a fluorescent tag to the capture oligonucleotide, which is detected by flow cytometry subsequent to hybridization to the bead populations. Two detection probes with different zip codes are required to cover each of the two possible alleles. This method has been successfully applied in the analysis of nine SNP markers located near the ApoE locus on chromosome 19.[8]

In OLA, one additional labeled reporter oligonucleo-tide is required for each variation. In minisequencing, only one primer for each allele is necessary, which is extended by one single-labeled nucleotide terminator. The SBCE primer, carrying a zip code at its 5'-dangling end, hybridizes to the target amplicon with its 3'-end one base upstream of the SNP query position. The primer is elongated by DNA polymerase using labeled dideoxynu-cleotide triphosphates (lacking a 3' hydroxyl group), so that only a single base—complementary to the polymorphic base being analyzed—is incorporated. The disadvantage of the SBCE format is that up to four different labels are required in multiplexing assays to cover each ddNTP, and hence a sophisticated flow cytometer with six fluorescence channels would be necessary. The alternative is to use a separate reaction for each type of terminator nucleotide. Then the analysis can be performed on a standard flow cytometer, but up to four different reactions are required in a multiplex assay to cover all possible nucleotides. This method has been successfully performed with 55 randomly selected SNPs near the ApoE locus.[9]

In allele-specific primer extension only one reaction is required for genotyping. Here, like in OLA, one oligonucleotide is designed for each allele to hybridize to the target amplicon with its 3' base complementary to the polymorphic base and a 5'-dangling end with a unique zip code. Standard deoxynucleotides are used, with one type of them (e.g., dCTP) being labeled. A DNA polymerase lacking 3'-5' exonuclease activity is used to elongate primers with a perfect match at their 3' end. During elongation, dependent on the sequence, several labels are incorporated which increases sensitivity compared to formats incorporating only one label (like OLA or SBCE). After the genotyping reaction, the zip-coded primers are hybridized to color-coded beads (Fig. 2). Only one fluorescent channel is required for detection of each possible genotype and no labeled oligonucleotides are required, which are expensive. This format seems to be the most suited and cost-efficient method for bead-based nucleic acid testing of SNPs and mutations. For evaluation, 20 randomly selected SNPs have successfully been analyzed in 633 probands applying this method.[10] A set of 100 color-coded beads carrying unique zip codes (FlexMAP Beads) is available as platform for the development of bead-based nucleic acid tests (Luminex). Several commercially available kits (Tm Bioscience, Toronto, Canada) have been developed with this platform, applying the ASPE format, including multiplex tests for screening of sequence variations in genes relevant for blood coagulation (Factor V, Prothrombin, MTHFR), drug metabolism (CYP450-2D6), and cystic fibrosis (CFTR).[3,11]

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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