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. 2005 Apr 28;33(8):e72.
doi: 10.1093/nar/gni071.

PieceMaker: selection of DNA fragments for selector-guided multiplex amplification

Johan Stenberg  1 , Fredrik Dahl, Ulf Landegren, Mats Nilsson
Affiliations

PieceMaker: selection of DNA fragments for selector-guided multiplex amplification

Johan Stenberg et al. Nucleic Acids Res. .

Abstract

We describe PieceMaker, a software tool for the design of applications of selector probes-oligonucleotide probes that direct circularization of target nucleic acid molecules. Such probes can be combined in parallel to circularize a selection of fragments from restriction digested total genomic DNA. These fragments can then be amplified in a single PCR using a common primer pair, yielding substrates for subsequent analyses, such as parallel genotyping or sequencing. However, designing multiplex selector assays is a laborious task. The PieceMaker program alleviates this problem by selecting restriction enzymes to generate suitable fragments for selection, and generating the output data required to design the selector probes.

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Figures

Figure 1
Figure 1
Illustration of definitions of design parameters. This Figure shows the definition of maximum flap length, fmax, and minimum and maximum selection length, smin and smax. The flap is the sub-fragment that is cleaved of by the structure-specific cleavage. The interval P of allowed positions for this cleavage is defined by fmax, smin and smax as well as by the presence of any possible restriction enzyme cleavage positions, none shown in this Figure.
Figure 2
Figure 2
The different cases of structure-specific cleavage position selection. The position of the region of interest (white) within the fragment (black) in relation to pmin and pmax determines the algorithm by which the cleavage position is selected.
Figure 3
Figure 3
In silico digestion. A target sequence is digested in silico in a reaction consisting of a single enzyme cleaving in the middle of the recognition sequence ‘TA’. This reaction is certain to cleave the target sequence at two positions; between positions 2 and 3, and between positions 17 and 18. Cleavage between positions 5 and 6 is possible, but not certain, since it depends on the nucleotide actually present at position 5. This digestion thus generates one fragment, having a length, l, of 15, with the region of interest beginning at position rb = 7 and ending at position re = 11. A possible restriction enzyme cleavage position cp exists at position 3. Selection of structure-specific cleavage position. Finding an optimal cleavage position, p, using the parameters b = ‘T’, smax = 15, smin = 5 and fmax = 10 next proceeds as follows: the interval P is [4, 10] according to the definition of P. Since pmin + 1 < rb < pmax, we have an instance of case (iii). Thus, we start searching for a ‘T’ in the sub-interval [4, 6]. Positions are interrogated in the order 6, 5, 4. As no ‘T’ was found, we continue the search in the sub-interval [7, 10], starting from position 7. We find a ‘T’ at position 8, and thus set p = 8. This gives C(p) = C(8) = 3.
Figure 4
Figure 4
Impact of parameter choice on design success rate. The x-axes show length limits for selected fragments; y-axes show proportion of targets for which the design was successful, in per cent. (A) The SNP target set. (B) The exon target set.
See this image and copyright information in PMC

References

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