Multiplex amplification enabled by selective circularization of large sets of genomic DNA fragments.
We present a method to specifically select large sets of DNA sequences for parallel amplification by PCR using target-specific oligonucleotide constructs, so-called selectors. The selectors are oligonucleotide duplexes with single-stranded target-complementary end-sequences that are linked by a general sequence motif. In the selection process, a pool of selectors is combined with denatured restriction digested DNA. Each selector hybridizes to its respective target, forming individual circular complexes that are covalently closed by enzymatic ligation. Non-circularized fragments are removed by exonucleolysis, enriching for the selected fragments. The general sequence that is introduced into the circularized fragments allows them to be amplified in parallel using a universal primer pair. The procedure avoids amplification artifacts associated with conventional multiplex PCR where two primers are used for each target, thereby reducing the number of amplification reactions needed for investigating large sets of DNA sequences. We demonstrate the specificity, reproducibility and flexibility of this process by performing a 96-plex amplification of an arbitrary set of specific DNA sequences, followed by hybridization to a cDNA microarray. Eighty-nine percent of the selectors generated PCR products that hybridized to the expected positions on the array, while little or no amplification artifacts were observed.
Dahl
Fredrik
F
Department of Genetics and Pathology, Rudbeck Laboratory Se-75185 Uppsala, Sweden.
Gullberg
Mats
M
Stenberg
Johan
J
Landegren
Ulf
U
Nilsson
Mats
M
D001483:Base Sequence; D004270:DNA, Circular / Q000737:chemistry*; D015894:Genome, Human; D023281:Genomics / Q000379:methods*; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D015345:Oligonucleotide Probes / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods*
D004270:DNA, Circular; D015345:Oligonucleotide Probes