Multiplex amplification of all coding sequences within 10 cancer genes by Gene-Collector

Abstract

Herein we present Gene-Collector, a method for multiplex amplification of nucleic acids. The procedure has been employed to successfully amplify the coding sequence of 10 human cancer genes in one assay with uniform abundance of the final products. Amplification is initiated by a multiplex PCR in this case with 170 primer pairs. Each PCR product is then specifically circularized by ligation on a Collector probe capable of juxtapositioning only the perfectly matched cognate primer pairs. Any amplification artifacts typically associated with multiplex PCR derived from the use of many primer pairs such as false amplicons, primer-dimers etc. are not circularized and degraded by exonuclease treatment. Circular DNA molecules are then further enriched by randomly primed rolling circle replication. Amplification was successful for 90% of the targeted amplicons as seen by hybridization to a custom resequencing DNA micro-array. Real-time quantitative PCR revealed that 96% of the amplification products were all within 4-fold of the average abundance. Gene-Collector has utility for numerous applications such as high throughput resequencing, SNP analyses, and pathogen detection.

Figure 1.

Principles of Gene-Collector. (A) A multiplex PCR is carried out using target specific primer pairs, generating both correct and incorrect products. For clarity, only three of the 170 primer pairs are shown and are color coded. (B) Guided by the collector probe, targets that contain matched primer pairs are circularized, leaving non-cognate products linear and thus susceptible to exonuclease degradation. In detail, (I) a collector probe contains complementary sequences to a cognate primer pair (orange). (II) The collector probe and the DNA ligase enable circularization of correctly amplified targets. (C) A universal amplification is then carried out using a randomly primed rolling circle amplification, generating a final product of concatemers of correct target sequences.

Figure 2.

Evenness measurements of the various stages of the Gene-Collector process assessed by quantitative PCR. A subset of 48 targets, all successfully amplified according to the resequencing array, was chosen to represent the overall variation in amplification efficiency. The starting material of human genomic DNA, assumed to be perfectly uniform, is compared to the evenness after the multiplex PCR, the ligation and exonuclease treatment and finally the rolling circle amplified material. The Y-axis represents a log-scale with deviations from 1 being relative differences from the average abundance. No compensation for differences in real-time PCR efficiency between reactions was used. However, the genomic DNA starting material represents a measure of this variation and the general imprecision of the real-time PCRs. Here, 96% of the final amplicons analyzed was no less than one-fourth of the average abundance.