Design of an FR primer.

<p>To a core fixed sequence (ATCTG), a series of Ns (A, T, G and C) are added in equimolar concentrations at each step of the oligo-nucleotide synthesis. This process generates all possible sequences of length n, when n bases are randomized, such that each variable sequence is attached to the end of the fixed sequence.</p

Multiplexed amplification of exons by splice signal FR primers.

<p>The FR primers for the donor (5′ splice signal) and the acceptor (3′ splice signal) splice sequences bind the exons with complementary base pairing over the entire length of the primers, by virtue of the presence of all the possible sequences within the randomized sequence portion of the FR primer. Only the specific primer molecule from the FR primer population is expected to bind selectively with full complementarity at the fixed sequence target site at a high Tm condition. The FR primers amplify multiple exons since they are capable of binding many exons within the human genome with complete sequence complementarity, wherever the fixed sequence binds.</p

Multiplex genome-wide exon amplification based on MYBPC3 gene exons 7 and 8.

<p>The human genomic DNA was PCR-amplified under standard conditions at 58°C with primers designed from the donor (5′) splice signal sequence (from exon 7) and the acceptor (3′) splice signal sequence (from exon 8) of the MYBPC3 gene. It was also amplified by the same FR primer pairs with decreasing number of fixed bases and increasing number of random bases (Ns) as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011138#pone-0011138-t001" target="_blank">Table 1</a>. The expected fragment (438 bases for the combined exon 7, intron 7 and exon 8) is present in all the lanes, and the number of fragments amplified increased with increasing Ns in the FR primers. M1 & M2 are marker lanes. Lane CS shows the computer simulated exon fingerprint obtained with the same primers used for lane 5, with four bases removed from the 5′ end of the forward primer and three bases removed from the 5′ end of the reverse primer (see text).</p

FR Primers used in the amplification experiments shown in Figure 4.

a<p>Excess FR primers required theoretically for each specific primer sequence within the primer population to be the same as in the standard PCR reaction compared to the actual excess used in the experiment.</p