Microarray synthesis through multiple-use PCR primer design

Abstract

A substantial percentage of the expense in constructing full-genome spotted microarrays comes from the cost of synthesizing the PCR primers to amplify the desired DNA. We propose a computationally-based method to substantially reduce this cost. Historically, PCR primers are designed so that each primer occurs uniquely in the genome. This condition is unnecessarily strong for selective amplification, since only the primer pair associated with each amplification need be unique. We demonstrate that careful design in a genome-level amplification project permits us to save the cost of several thousand primers over conventional approaches.

Minimum Multicolored Subgraph Problem in Multiplex PCR Primer Set Selection and Population Haplotyping

“…Since the efficiency of PCR amplification falls off exponentially as the length of the amplification product increases, an important practical constraint is that the two primer sites defining a product must be within a certain maximum distance L of each other. In applications such as spotted microarray synthesis [1] a further pairwise compatibility constraint is the requirement of unique amplification: for every desired amplification locus there should be a pair of primers that amplifies a DNA fragment surrounding it but no other fragment.…”

“…As noted by Fernandes and Skiena [1], primer selection problem subject to pairwise compatibility constraints can be easily reduced to M(W)MCSP: each candidate primer becomes a graph vertex and each pair of primers that feasibly amplifies a desired locus becomes an edge colored by the respective locus number. More generally, the problem of selecting the minimum size/cost set of primers required to amplify at least k of the n loci reduces to M(W)kCSP; this problem arises when several multiplex reactions are required to amplify the given loci.…”

“…Fernandes and Skiena [1] studied MMCSP with at most one color per edge in the context of multi-use primer selection for synthesis of spotted microarrays.…”

The Parameterized Complexity of the Rainbow Subgraph Problem

“…Note that in the worst case, this reduction might not produce a polynomial-size instance, as the number of possible explanations of each genotype may become exponential. Another bioinformatics application appears in the context of PCR primer set design [1,3].…”

Effective heuristics for the Set Covering with Pairs Problem

“…PCR requires the presence of two single-stranded DNA sequences called primers, which complement specific parts of either the forward or reverse strand of the double-stranded DNA and enable duplication of the region in between. Heuristics and approximation algorithms for this problem were developed in (Fernandes and Skiena, 2002) and (Hajiaghayi et al, 2006). Hermelin et al (Hermelin et al, 2008) developed approximation algorithms for the Minimum Substring Cover Problem, where the elements to be covered are strings in S and the elements to cover them are their substrings.…”