Info

Silane

Semicarbazide

[33]

Silane

Thiol

[34]

The second choice is whether or not to treat the substrate surface to further enhance the binding of the macromolecule or substance. Table 2 lists a variety of modifications which have been used and range from simply coating with poly-lysine[4] or using a multistep procedure with polyethylenimine.[25] The most common method is silanization and several silane derivatives are listed in Table 2. Some unique slide coatings are the use of microporous polymer, agarose file, and a deposition of gold substrates. Glass slides coated with amine, aldehyde, epoxy, thiol, or gold derivatives as well as a plain silylated form (CEL slides) are commercially available through TeleChem International, Inc.[26]

The third choice is a form of macromolecule to print. In the past, some investigators chose to print whole bacterial plasmids containing the cDNA insert.[1,13] More commonly today, cDNA is printed started with its double-stranded purified form. Recent references refer to the printing of long vs. short oligos; short oligos being 25-mers or less and long oligos being 40-mers or longer. Although factors, such as GC content and overall sequence similarity, are important and need to be considered for designing oligos to be printed,[35] several references have shown long oligos to be comparable in sensitivity and specificity to cDNA probes.[36,37]

Once the type of macromolecule is chosen, the quality of the printed macromolecules should be checked. One method is to check the PCR products (cDNA probes) by agarose gel electrophoresis. Any product which shows more than one band or does not show any product at all is not arrayed and can be substituted with a ''cleaner'' alternative product corresponding to the same gene. Also,

Fig. 2 Experimental prototype of a DNA Microarrayer: a Hamilton Microlab 2200 (Reno, Nevada) modified with a 32 Stealth printhead and SMP3 Micro Spotting Pins (TeleChem International, Inc., Sunnyvale, CA). Metal platters (Machine shop at HARC) have been fashioned to hold five slides during attachment, and will be set on top of the platform. The platform is capable of holding one microtiter plate and seven of these platters, allowing 35 slides to be printed in each round. (View this art in color at www.dekker.com.)

Fig. 2 Experimental prototype of a DNA Microarrayer: a Hamilton Microlab 2200 (Reno, Nevada) modified with a 32 Stealth printhead and SMP3 Micro Spotting Pins (TeleChem International, Inc., Sunnyvale, CA). Metal platters (Machine shop at HARC) have been fashioned to hold five slides during attachment, and will be set on top of the platform. The platform is capable of holding one microtiter plate and seven of these platters, allowing 35 slides to be printed in each round. (View this art in color at www.dekker.com.)

it is necessary to obtain an accurate quantification of the molecule to be arrayed. Yue et al. describes using an assay with PicoGreen.[38]

The fourth choice is whether to modify the macromol-ecule. Most cDNA probes are modified by adding an amino group at either the 3'- or 5'-ends.[4'12'19'20] Other modifications have included thiol,[10'28'31] disulfide,[34] or benzaldehyde groups;[33] although unmodified DNA probes have shown equal success.[32,37] In addition, some investigators have used chemical linkers to connect the modified surface with the modified probe. Yang et al. used a maleic anhydride linkage between a thiol-modified oligo and an aminosilane-modified surface.[31]

The fifth choice is the type of printing method to use. Printing methods have been divided into contact and noncontact methods.[39,40] The contact method uses mi-crospotting devices to deliver the macromolecular solution to the printing surface. An example is depicted in Figs. 2 and 3. These devices deliver the solution by capillary action and tips are either disposed of or washed thoroughly before the next printing. Initial devices were hand-built using an xyz-axis gantry robot with the macromolecular solutions being delivered through stainless steel printing pins (Stanford University, National Institutes of Health). Pin heads are now available in ceramic (Apogent Discoveries, Hudson, NY) and tungsten (Point Technol ogies, Boulder, CO). Other delivery methods are capillaries, tweezers, or quills, and Holloway et al. describes several commercially available arrayers.[40]

Noncontact methods include inkjet printers which disperse solutions of macromolecules onto the array surface without the dispensing tool touching the array surface. Many of these devices use piezoelectric technology where an electrical current controls droplet formation to precise measurements.1-19,41-1 Two current commercial vendors include Shimadzu Biotech (Pleasanton, CA) and PerkinElmer (Boston, MA).

After printing, several investigators recommend treating the printed surface to ensure a tight covalent bond and to lessen nonspecific binding during hybridization. The most common method is to cross-link the macromolecules to the surface with UV light.[32,37] This method has been used with glass slides as well as nylon filters. Other protocols bake the slides in a vacuum oven[4] and/or treat with succinic anhydride[4] or just treat with a mixture of succinic anhydride and acetic anhydride.[42]

Finally, it is recommended that a set of protocols be instituted to assess the quality of the printed slides. Microscopic examination of the slides with food coloring or hybridizing the slides with a labeled PCR primer (used to make the arrayed PCR products) has been used with random sampling, but these agents can destroy the active sites on the probes.[43] Other investigators have successfully used nondestructive means to assess quality, such as dCTP-Cy3 or SYBR Green dyes.[44,45]

Fig. 3 Experimental prototype of the DNA microarrayer: a close up view of a 32-pin dry station (Die-tech, San Jose, CA) that has been mounted to a specially designed wash station (Machine shop at HARC) secured to the base of the MicroLab 2200 between the two platforms. (View this art in color at www.dekker.com.)

Fig. 3 Experimental prototype of the DNA microarrayer: a close up view of a 32-pin dry station (Die-tech, San Jose, CA) that has been mounted to a specially designed wash station (Machine shop at HARC) secured to the base of the MicroLab 2200 between the two platforms. (View this art in color at www.dekker.com.)

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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