Application of Circular Ligase to Provide Template for Rolling Circle Amplification of Low Amounts of Fragmented DNA

Compromised biological evidence, particularly those samples that contain a very small amount of DNA or may have a significant amount of degradation, may not yield a complete or useful DNA profile. One approach that shows promise to overcome the limited quantity of DNA issue is the use of Whole Genome Amplification (WGA). The WGA method amplifies all the DNA in a sample, yielding higher levels of DNA template that can be subsequently analyzed using standard forensic technologies. One WGA technique termed Rolling Circle Amplification (RCA) involves the amplification of circular DNA fragments such as those of plasmids and viral genomes. With a highly processive polymerase, RCA can yield microgram quantities of DNA from circular templates. However, with the exception of intact mitochondrial DNA (mtDNA), circular DNA fragments do not occur naturally in humans. Consequently, RCA would have limited application as would most WGA methods for fragmented or degraded DNA evidence. However, a commercially-available circular ligase enzyme (CircLigaseTM, EPICENTRE Biotechnologies) which possesses a catalytic activity that circularizes single-stranded DNA (ss-DNA) offers the potential for RCA-WGA to be used on compromised DNA materials. This paper describes a study focused on optimizing the circularization reaction of several DNA templates, including ss-DNA and denatured double-stranded DNA (ds-DNA). In addition, a detection method was developed to identify the circular products of various templates in order to monitor optimization as circular DNA ladders are not commercially available. Optimization studies involved varying experimental conditions such as incubation time and concentrations of manganese chloride, ATP, and enzyme. Results show that the optimal manganese chloride concentration varies depending on the size of the template. However high concentrations (5 mM) of manganese chloride appears to induce nonspecific degradation of the template. Neither higher than manufacturer’s suggested ATP concentrations nor increased incubation periods yield any discernible benefit or detriment to circularization. However, higher circular ligase concentrations appear to increase the yield of circular products. Templates with different terminal nucleotides were used to examine the efficiency of circularization, and the results demonstrate that the efficiency of circularization is dependent on the specific base present at both the 5' and 3' terminal ends. The activity of the enzyme using denatured, ds-DNA template was also examined. The methods described should form the foundation for developing protocols to circularize human DNA fragments that would be suitable for RCA. Thus, it may be possible to obtain partial or perhaps complete mtDNA and/or nuclear DNA profiles from compromised forensic evidence. Introduction: DNA has many characteristics that make it a robust material for individual identification. However, DNA evidence can be degraded to the point that analysis of the samples is challenging or even impossible. Whole Genome Amplification (WGA), a novel method that increases the concentration of high quality templates may overcome the aforementioned problem. However, WGA is most successful when using high quality template and can fail to yield sufficient representative amplified DNA when the initial template is highly degraded (1). However, Rolling Circle Amplification (RCA) and Multiple Displacement Amplification (MDA), variations of WGA, offer the potential to yield results even from highly degraded samples (2). Both techniques use Ф29 polymerase, an enzyme with a low error rate (1 in 10 bp) when amplifying DNA (3, 4 and 5). The MDA and RCA methodologies are based on amplification of any template using degenerate primers. But the RCA technique exploits the efficiency of the polymerase with circular templates. Circular templates in essence are infinite length templates and thus are not as refractory to WGA as linear degraded template DNA. Since circular template, other than intact mtDNA, are not typically found in the human genome, other techniques have been applied to achieve template circularization so that RCA can be used effectively. One of these techniques uses T4 DNA ligase. This enzyme forms phosphodiester bonds between the 5' phosphate and the 3' hydroxyl groups of ds-DNA. However, the enzyme will ligate dstemplate forming concatamers; the ligation of ds-DNA is for the most part inefficient and the ligation of sstemplate is very poor (6). Because of these features subsequent analyses are problematic with T4 DNA ligase treated templates. Epicentre Biotechnologies offers a new enzyme called CircLigaseTM that circularizes single stranded DNA. This enzyme ligates the template by the 5'phosphate group and the 3' hydroxyl group. It circularizes fragments larger than 30 nucleotides and up to 1.9 kb. Use of CircLigase can provide circular products from ss-DNA template for RCA; however its activity on ds-DNA template is unknown (7). Because this is a novel technique, optimization studies need to be performed. Materials and Methods: Circularization Assay Templates used were synthetic, phosphorylated oligonucleotides with known sequences and sizes (Integraded DNA Technology (Coralville, IA), PAGE purified). The DNA final concentration for the circular ligase reaction was 0.5μM, the amount recommended by the manufacturer. Ligation was performed according to the manufacturer’s recommendations. Master mix for samples treated with CircLigaseTM was composed of 1X reaction buffer, 50μM ATP, 2.5 mM MnCl2, 5 Units/μL of CircLigaseTM (Madison, WI) in a final volume of 20μl. For untreated samples, the enzyme was substituted with deionized water. The positive control provided in the kit was run with all samples. After ligation, samples were treated with exonuclease I or III (Exo I, Exo III), enzymes that digest ssand ds-DNA respectively (8 and 9). For the exonuclease digestion, 10μl from the 20 μl circular ligase reaction were taken and treated with 1μl of exonuclease and the remaining 10μl of the reaction were mixed with 1μL of molecular grade water.