Experimental

Genotyping by ASO applies to previously ascertained polymorphisms and/or mutations and is not intended to discover new DNA variants. Through PCR amplification, one aims at a single polymorphic site, but it is also customary to target a number of variant sites residing within a longer amplifiable DNA segment[1] or even those scattered on unrelated genomic fragments that can be simultaneously amplified in a multiplex PCR.[2]

Following PCR, amplicons are immobilized on a solid support by dot blotting and ultraviolet (UV) fixation,[3] usually in duplicates to allow parallel probing of both alleles with the corresponding ASO probes. Typical result of ASO hybridization is illustrated in Fig. 1. SNPs are usually biallelic; even if they are not, the same detection principles apply to triallelic sites or small haplotypes composed of two polymorphic sites, one next to another, except that more than two probes could then be needed. Usually ASO probes are designed as 15-mers to 20-mers, with the polymorphic site in the middle of the sequence and not less than three nucleotides from the end. The shorter is the oligonucleotide probe, the more pronounced is the effect of mispairing at the polymorphic site, thus facilitating discrimination between the alleles. However, an overall duplex stability counts as well; too short a probe could render a signal of a fully matched complex unstable and compromise its detection. In our hands, 15-mer ASO probes worked well, whereas more than 20-mers were also recommended by others.

In a classical ASO protocol, great emphasis is made on the choice of the hybridization temperature to discriminate between fully matched and mismatched probes.[5] Hybridization is carried out a few degrees below the melting temperature (Tm) of the fully matched probe. In practice, the specificity (i.e., discrimination between the alleles) is achieved through posthybridization washes that selectively eliminate the mismatched, less stable probe from the target. The washing temperature, time, and salt concentration are experimentally determined and are typically ''personalized'' in each laboratory. In an alternative dynamic ASO protocol,[6] rather than using constant temperature, the hybridization step covers a temperature gradient, from above the melting point down to room temperature (75-30°C), such that each probe has an opportunity to find its optimal hybridization conditions. To prevent the formation of the mismatched complex between the labelled probe and the opposite allele, the unlabelled opposite oligonucleotide is added in 15-fold or greater excess to the hybridization mixture. Such blocking of the mismatched binding by using the opposite allele probe as a competitor has been used before.[7,8] It appears that the dynamic ASO makes full use of the presence of the competitor. The limitation in

Fig. 1 ASO hybridization of G/A polymorphism at position 80 in RFC1 (reduced folate carrier) described earlier by Laverdiere et al.[4] The PCR product of 207-bp encompassing G80A polymorphic sites was transferred in duplicate to a HYBOND N+ (Amersham Pharmacia Biotech) membrane. Identical twin membranes were hybridized with the ASO probe specific for the G80 (5' gcacacgaggCgccg) or A80 (5' gcacacgaggtgccg). Hybridization with ASO-specific probe was carried out at 45°C in a x 20 excess of the nonlabeled probe for the other variant allele.

Fig. 1 ASO hybridization of G/A polymorphism at position 80 in RFC1 (reduced folate carrier) described earlier by Laverdiere et al.[4] The PCR product of 207-bp encompassing G80A polymorphic sites was transferred in duplicate to a HYBOND N+ (Amersham Pharmacia Biotech) membrane. Identical twin membranes were hybridized with the ASO probe specific for the G80 (5' gcacacgaggCgccg) or A80 (5' gcacacgaggtgccg). Hybridization with ASO-specific probe was carried out at 45°C in a x 20 excess of the nonlabeled probe for the other variant allele.

the probe design is that both of them have to be from the same strand, ideally of the same sequence and differing only at the polymorphic site. However, the ASO for one allele serves, at the same time, as the competitor for the second allele, and vice versa. Following hybridization, dot blots are removed from the solution, rinsed at room temperature to wash out excess nonbound radioactivity, and exposed. No additional specificity-increasing washes are required. The most important advantage of dynamic ASO is that the same hybridization conditions work for most of the systems. As a consequence, multiple hybridizations can be simultaneously carried out in the same hybridization oven, disregarding different Tm values of the ASO probes used.

Hybridization

Membranes are prehybridized at 75°C for 30 min (Boekel Big Shot hybridization oven) in 25 mL of 1 x SSPE [150 mM NaCl, 10 mM NaH2PO, 1.1 mM EDTA, pH 7.4, 0.75 M NaCl, 70 mM Tris/HCl, pH 7.4, containing 1% sodium dodecyl sulfate (SDS) and 200 mg/mL heparin]. ASO probe (15-mer) is 5'-labeled using g-[32P]-ATP (6000 Ci/mmol; Amersham Pharmacia Biotech) and T4 kinase (Gibco BRL). Usually the volume of hybridization mixture is increased by 5 mL for each additional membrane. The ASO probe is added to the hybridization mixture to a final concentration of 50 pM accompanied with the opposite allele competitor at 750 pM. Hybridization is then carried out at a decreasing temperature by letting the oven cool down to 30°C and leaving 30 min for equilibration at this temperature. After two brief washings in 2 x SSPE containing 1% SDS at room temperature, the membranes are exposed at — 80°C with intensifying screens or read by Phosphoimager. Most of the polymorphisms do not require any special adjustment of this protocol and work outright. If the background appears too high and simple shortening of the time of exposure does not help, it can be eliminated: 1) by increasing the competitor/probe ratio; 2) by increasing the probe concentration (keeping the same competitor/probe ratio); or 3) by decreasing the amount of dot-blotted target DNA. Rarely, design of new ASO probes is required.

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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