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WIREs Forensic Sci

Rolling circle amplification: A (random) primer on the enrichment of an infinite linear DNA template

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Abstract Rolling circle amplification (RCA) is a robust enzymatic process in which a circular DNA molecule serves as a template for exponential amplification of molecular targets of interest. Random primers that have bound to DNA sequences across the circularized DNA template initiate copying via the action of a highly processive DNA polymerase. Following a cascade of strand displacement events, long tandem copies of the original template sequence are generated. Because the template molecule is in a circular format, it essentially serves as an infinite linear template. In conjunction with selective enrichment techniques, RCA may prove highly useful in the selectivity and sensitivity of current DNA typing systems. In addition, circularization potentially may allow for amplification of fragmented and low copy number DNA encountered in forensic casework samples that can be subsequently typed. This article is categorized under: Forensic Biology > Forensic DNA Technologies
Illustration of rolling circle amplification technique. (a) Random (degenerate) primers (blue) first bind to a circular DNA template molecule (green) and a DNA polymerase (black) associates with the primers to begin primer extension. (b) Nucleotides are continuously incorporated into the growing DNA strand by a DNA polymerase to generate a copy of the original DNA template (purple). (c) Eventually, the DNA polymerase will encounter a region where another primer had previously annealed. The growing strand displaces the strand in front of it and continues to extend. (d) Due to a cascade of these strand displacement events, long tandem copies of the original DNA template are generated (Polidoros et al., )
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Illustration of padlock probe targeting and circularization followed by RCA. (a) The target‐specific hybridization arms of the padlock probe bind to the DNA template flanking the SNP. (b) A DNA polymerase (without strand displacement activity) fills the gap in a 5′→3′ direction incorporating the complementary state of the SNP target into the sequence of the padlock probe. The padlock probe is ligated to form a circular molecule and RCA degenerate primers hybridize to initiate the amplification process. (c) RCA replication yields linear tandem copies of the padlock probe containing the target of interest (Lizardi et al., )
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Diagram depicting the restriction and circularization‐aided rolling circle amplification (RCA‐RCA) process. (a). DNA containing damaged sites (yellow) is cut at restriction sites (black triangles) to yield short DNA fragments (b). (c) These digested fragments either self‐ligate or cross‐ligate to produce circular molecules and any remaining linear DNA is digested. (d) Intact circular molecules allow for exponential amplification via RCA whereas synthesis is halted in strands containing damaged DNA sites. (e). Circular templates are subjected to RCA (Wang et al., )
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