Multiplex amplification enabled by selective circularization of large sets of genomic DNA fragments

Nucleic Acids Research
Fredrik DahlMats Nilsson

Abstract

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 microarr...Continue Reading

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Related Concepts

Genome
Nucleic Acid Hybridization Procedure
Complex (molecular entity)
Digests
Gene Amplification Technique
Genomics
Oligonucleotide Primers
Protein Domain
Oligonucleotides
Oligoribonucleotide Probes

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