Multiplex allele-specific target amplification based on PCR suppression

Abstract

We have developed a strategy for multiplex PCR based on PCR suppression. PCR suppression allows DNA target amplification with only one sequence-specific primer per target and a second primer that is common for all targets. Therefore, an n-plex PCR would require only n ؉ 1 primers. We have demonstrated uniform, efficient amplification of targeted sequences in 14-plex PCR. The high specificity of suppression PCR also provides multiplexed amplification with allele specificity. Multiplexed PCR was used to develop assays for genotyping DNA samples from cystic fibrosisaffected individuals. The new approach greatly simplifies primer design, significantly increases the PCR multiplexing level, and decreases the overall primer cost. In addition, this assay is more readily amenable to automation and is therefore suitable for high-throughput genetic diagnostics.

The evolutionary-developmental analysis of plant microRNAs

“…The first step of this procedure consists of the polymerase chain reaction (PCR) suppression -amplification (Broude et al 2001) of miRNA gene fragments from adaptor-ligated plant genomic DNA using an miRNA-specific PCR primer in combination with an adaptor primer that is common to all DNA molecules. Such amplifications typically yield mixtures of PCR products containing a proportion of miRNA gene fragments.…”

“…These amplifications were performed on further aliquots of adaptor-ligated genomic DNA by the sequential use of two miRNA gene-specific primers derived from the novel sequences obtained, in combination with the A-adaptor primer (Broude et al 2001). The resulting PCR products were cloned in plasmid vectors and sequenced, and the novel data obtained were used to complete the sequences of the putative MIR164 genes identified.…”

“…Genomic DNA of Z. mays inbred line A188 (Gerdes & Tracy 1993) was kindly provided by Dr Peter Rogowsky (RDP, Lyon). To amplify miRNA gene sequences, PCR-suppression amplifications were performed, based on the method of Broude et al (2001). Aliquots of genomic DNA (10 mg) from all species studied were digested with DraI or HaeIII.…”

“…Double-stranded DNA adaptors (Broude et al 2001) were ligated to the resulting cleaved DNA fragments, and unincorporated adaptors were then removed using NucleoSpin PCR clean-up columns (Macherey-Nagel). PCR amplifications were performed from aliquots (25 ng) of adaptor-ligated DNA, using a 21-nt primer corresponding to the consensus miR164 sequence from A. thaliana (figure 1) and the A-adaptor primer (Broude et al 2001). Amplifications were also performed using a 19-mer miR164 primer, lacking the two bases at the 3 0 -extremity of the consensus miR164 sequence.…”

Multigene amplification and massively parallel sequencing for cancer mutation discovery

“…However, one of the crucial problems with PCR is that when large numbers of specific primer pairs are added to the same reaction, undesired amplification products arise (15). Even with a careful primer design, PCR usually is limited to 10 simultaneous reactions before amplification yield is compromised by the accumulation of irrelevant products (16,17).…”