Microarray Armspcr

The advent of microarray technology promises to deliver rapid, high-throughput genotyping through the development of solid-phase, miniaturized assay formats. The forerunner of microarray ARMS-PCR came in the form of allele-specific primer extension, where arrayed allele-specific primers are queried with multiplex preamplified template DNA containing the polymorphisms of interest in the presence of reverse transcriptase and a mixture of ribonucleotides, one of which is fluorescently labeled. Practical difficulties in performing solid-phase PCR, as

Table 1 Alternative designations for ARMS-PCR

Method designation

Reference

Mutation-specific PCR [1]

Amplification refractory mutation [2] system (ARMS)

Allele-specific PCR (AS-PCR) [3-5]

PCR amplification of specific alleles (PASA) [6]

PCR with sequence-specific primers (PCR-SSP) [39]

well as the markedly higher signal-to-noise ratio achieved with RT, were the principal reasons for its use here instead of DNA polymerase. These limitations were recently overcome with the development of a two-phase, on-chip PCR platform that achieves simultaneous multiplex amplification of genomic DNA and immobilized allele-specific

primer extension using Taq DNA polymerase.

Design and Optimization of ARMS-PCR Assays

With its numerous formats, ARMS-PCR offers unparalleled flexibility in the design of genotyping assays. Major initial considerations guiding the design process itself include the number and type of genetic variation(s) under study; available equipment, personnel, and expertise in primer design; desired sample throughput; and cost. Once a suitable ARMS-PCR format has been selected, the issue of allele-specific amplicon detection should be separately addressed (reviewed elsewhere in this volume). The design process is complete following customization of the assay to the polymorphisms of interest (primer design), optimization of PCR conditions, and troubleshooting of any problems.

Optimization and Troubleshooting

For all its merits, ARMS-PCR is subject to the same limitations that apply to conventional PCR. Problems such as mispriming and primer dimer formation can be minimized by using suitable software for basic primer design. Complications in basic primer design arising from polymorphisms lying close to the SNP of interest can be overcome by strategically incorporating deoxyinosine (dl) residues into the primer sequence.[1]

Since the original description of ARMS-PCR, several authors have shown that primer 3'-terminal mismatch discrimination with DNA polymerases is not absolute; extension of such mismatches does occur, albeit at a much lower rate than that of a perfectly matched primer. Extension efficiency depends on several factors, primarily the nature of the mismatch[4,20-24] as well as its sequence context,[23] template abundance,[25] and dNTP concentration.1-1,21-1 DNA-dependent DNA polymerases generally achieve better allelic discrimination than reverse tran-scriptases.[21,22]

While optimization of the PCR reaction itself is typically straightforward, the potential for discrimination failure occasionally necessitates troubleshooting of ARMS-PCR. Initial measures should focus on readily modifiable reaction components and might include reducing the number of amplification cycles,[24] the amount of template DNA,[25] dNTP concentration,1-1,8-1 or performing the reaction under rapid-cycle conditions.[26] In rare instances where these measures would fail or prove difficult to implement (e.g., when running multiplex reactions or pooled DNA),

Table 2 Comparison of different platforms for ARMS-PCR

Method designation

Format

Special features

Reference

ARMS-PCR (AS-PCR; PASA)

Two tube

[1-6]

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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