CICEET Progress Report for the period 9/16/06 Through 3/15/07

Project Title: High-throughput Quantitative Detection of Microbial Contaminants
Principal Investigator(s): Mara R. Diaz
Additional Investigator(s): Kelly Goodwin and Jack W. Fell
Project Start Date: Nov 2006 to March 2007

Figures

Figure 1

Figure 2

Figure 3

Figure 4

Tables

Table 1

Table 2

Several benchmarks were established for this “bi-annual” report. However, given the circumstances that funding for this project and money for expenditures were not available until mid November 2007, rather than September 1st, this progress report covers ~4 months of work (Nov 15 - Feb 5) and therefore does not fully reflect the benchmarks that were aimed for the first 6 month period.

The immediate objectives for this term included:
i) Probe design and validation.
ii) Optimization of ligation and exonuclease reaction.
iii) Optimization of amplification system

Tasks to meet objectives
i) Padlock Probe design and validation. A nucleotide sequence alignment (DNAstar Megalign), which consisted of over 100 dinoflagellate species, was constructed to facilitate probe selection for the identification of K. brevis and K. mikimotoi. This database, which is comprised of in-house and public domain sequences, represent sequences of the D1/D2 of the Large subunit rRNA gene. Sequences were Align with CLUSTAL W. Areas displaying sequence divergence among the species were selected and analyzed using the software program Oligo (Molecular Biology Insights, Inc). The padlock probes, which consisted of 96 to 98 mers, were designed to contain two target complementary sequences separated by a universal primer coding region that facilitates amplification and a zipcode sequence that acts as a sequence identifier for the detection system. For specific probe recognition, the probes are designed to bear the discriminatory bp in the 3’end.

The parameters to establish the quality and thermodynamics of prospectus probe sequences included: hairpin structures, Tm by the nearest neighbor algorithm, primer dimer and free energy of reaction. The probes were validated with GenBankBlast to assure the overall probe specificity. After successful validation, the prospectus probe was tested with synthetic complementary oligonucleotide sequence. This step was followed by testing the probe sequence with fragmented DNA.

ii) Optimization of Ligation reaction and Exonuclease reaction. Optimization of ligation reactions were assessed empirically by testing different concentrations of enzyme, target DNA and probe concentrations.

The specificity of the ligation reaction was validated with synthetic target DNA that bears single mutation at different locations of the probe sequence, specifically at the 3’end of the probe.

Cycling conditions were also tested at different annealing temperatures. The reactions were performed in sufficient quantities to allow analysis on agarose gels. Gel electrophoresis allows the identification of unsuccessful ligation or possible cross-reactivities with non-target DNA.

Exonuclease reaction. The assay was conducted with exonuclease I and exonuclease III. The assay was undertaken at various enzyme concentrations and at different time intervals to determine the required time needed to eliminate unbound probes.

iii) Optimization of Amplification system. To verify the optimization of ligation and exonuclease conditions, the ligated samples were subject to PCR amplification and further detected using gel electrophoresis. Amplicons were tested before and after the exonuclease treatment to determine the extent of unreacted or dimerized probe.

Progress on Tasks
i) Probe design and validation. Five different species-specific probes were designed and computer validated (Table 1). Among this list, K. brevis probes, Pkb(a) and PK(b) were tested with synthetic target and non- target sequences.

ii) Ligation. Different probe concentrations were tested to find the optimum concentration. To meet this task, serial dilutions of PLPs were tested with a fixed concentration of target DNA (10nM).

Detection limits of the ligation reaction were undertaken with serial dilutions of synthetic target DNA and corroborated via PCR and by resolving the amplicon product with gel electrophoresis. The next step is to determine the detection levels with K. brevis genomic DNA.

The specificity of the ligation reaction is currently being examined with a series of synthetic target oligonucleotides bearing a single mutation at different locations in the 3’ end of the probe.

iii) Exonuclease treatment. The optimum exonuclease conditions were determined. However, we are currently exploring the use of higher enzyme concentrations in order to shorten the incubation time.

iv) Amplification system. In order to verify the outcome of ligation and exonuclease conditions, optimization of certain PCR parameters ie. amount of target, number of cycles and various annealing temperatures were tested.

Have the results/data gathered during this reporting period changed the project objectives when compared to your original proposal?
The main objective of the project has remained the same.

Dissemination activities during this reporting period
CICEET Workshop ­ Jan. 17, 2007 - Alabama
Article: “Inside R & D and Alert” - Technical Insights Frost and Sullivan. Jan 2007

Difficulties
Probe design. The designing of Padlock probes can pose some challenges. These challenges are mainly attribute to complex structural conformations consisting of hairpin loops, stems and loops structures. These secondary structures not only can hamper hybridization efficiency but reduce the flexibility in oligonucleotide probe design. For instance, the first probe designed to target K. brevis (PK(a) did not give any signal when challenged with genomic DNA or its complementary synthetic target sequence. After numerous in vitro attempts, the probe sequence was re-analyzed using another software program ie McFold. To our surprise, this probe sequence suffered from potential secondary structures in the 5 and 3’end, which were not detected with the Oligo software program. Based on this experience we will implement the use of both software programs when designing prospectus sequences.

Another current difficulty is the specificity of the assay, which at the time of this report is not meeting our expectations. Toward this end, we are experimenting with different ligation conditions such as temperatures and buffer systems.

Data Generated to date
Probes design and validation. In order to have a better control of a working model for prospectus probe sequences, we shifted our protocol strategy by testing the prospectus probe with synthetic mers. This strategy, which allows better optimization of assay conditions, will be followed by challenging the probe with genomic DNA. Therefore, the results herein presented were mostly obtained with synthetic target sequences.

Our first prospectus probe sequence, Pk(a) failed to ligate with our fragmented target DNA (genomic DNA). Similar results were obtained when the probe was tested with a synthetic complementary oligo. As explained in Section F. failure of ligation can be attributed to secondary structural conformations that interfere with the duplex formation between the target and the probe. The latter was confirmed with MFold, a software program that predicts secondary DNA structures using nearest neighbor thermodynamics. In view that this probe sequence failed to hybridize with its target, this probe was redesigned using different sequences in the backbone area. Modification to this region resulted in a probe binding site free of potential secondary structures and allowed efficient ligation between the probe (Pkb) and synthetic target.

The ligated product was tested under different PCR conditions using 30, 35, and 40 cycles. When the ligated products were amplified at 50°C, a smear banding pattern was observed (Figure 1). This smear pattern was abolished by decreasing the probe concentration, reducing the number of PCR cycles, and by increasing the annealing temperature to 55°C (Figure 1). These changes produced a series of amplicons or a concatemer of bands that were eventually eliminated by adding to the ligated product exonucleases I and III (Figure 1). A single product representing the correct amplicon size indicated the success of the ligation reaction. To optimize the exonuclease reaction, different incubation times ranging from 45 min to 1hr were employed (Figure 2). Although no spurious PLP amplicons were observed after 15 min incubation, one hr incubation was selected to assure the removal of any non-ligated reactants.

In order to further optimize ligation conditions, different PLP concentrations were tested. The concentration ranged from 400pM to 5pM. Beyond 5 pM the signal was barely detectable in the agarose gel. Since high concentrations of probe can lead to background problems, especially at concentrations ranging from 400 to 100 pM, is of uttermost importance to determine the optimum probe concentration for each probe.

Detection Limits. To establish the detection limits of the ligation reaction, we tested various artificial template concentrations ranging from 500 pM to 50 fM. After the exotreatment, aliquots of the exo product were subject to PCR reactions. Figure 3 depicts the agarose gel electrophoresis of one of the experiment conducted at concentrations ranging from 100 to 50 fmol. The results showed that concentrations as low as 500 fmol can be detected when using PCR as the detection system. Beyond that concentration the signal is barely detectable (Figure 3).

Specificity. Kbrev(b) probe was challenged with its complementary synthetic target and non-target oligonucleotides bearing single base pair mismatches at different locations of the 3’arm or 5’end of the probe (Table 2). Among all the non-targert sequences, RC-C was selected as the only representative sequence bearing a mismatch at the 5’end. Other locations within the 5’end were not tested since mismatches at 5’end arm do not provide good discrimination. The aim of this experiment was to determine the level of specificity that can be attained with a single base pair mismatch and how the mismatch position can affect the specificity of the assay. Ligation conditions were initially carried out at 55°C. Under these conditions all target and non-target sequences amplified. However, based on the electrophoresis results, RC-B showed the weakest band among all the non-target sequences (Figure 4). This synthetic oligo bears a mutation at the ligation junction on the 3’end. In contrast, R/C-C, produced a brighter band. These results are not surprising as better discrimination and fidelity conditions are achieved when single mismatches are located at the 3’end. As expected, mismatches located away from the ligation point did not provide good level of discrimination as they all amplified. In view, that the ultimate goal is to have “zero” tolerance for cross reactivity, especially for mismatches at the 3’end of the ligation junction, we tried to increase the stringency reaction by increasing the ligation temperature. However, temperatures as high as 65°C did not meet our expectations. Other attempts, such as the inclusion of an excess of non-related DNA have been assessed. However, RC-B continues to amplify. On the other hand, inclusion of TMAC buffer, which is known to increase the stringency of the reaction, proved too stringent. At this point, several other strategies are being explored.

Digestion analysis. Enzymatic target preparation was undertaken with K. brevis. Toward this end, 350 ng of genomic DNA was digested with different restriction enzymes ie. EcoRI, HindIII and MboII. These reactions were undertaken with the corresponding supplied buffers and manufacturers protocol. The enzymes were tested individually or combined. Based on the electrophoretic pattern, MboII was found to be effective at cutting K. brevis DNA (Fig 5). This enzyme recognizes the sequence GAAGA (8/7). Alw26I, which recognizes the five base pair sequence GTCTC, was also tested and yielded a successful digestion (data non shown). Both enzymes recognition sites are located at position 626 and 282 of the large subunit ribosomal rRNA gene.

Project Objectives for Next Reporting Period

Objectives
i. Improve ligation assay specificity
ii Evaluation of current assay conditions with target and non-target genomic DNA
iii Development of assay conditions for rolling circle amplification

Work plan to Meet Objectives
Attempts will be made to improve the discrimination of single bp mismatch at the point of ligation. This task, which is currently underway, will explore the incorporation of other buffer systems with higher anionic content. In addition, the temperature of ligation conditions will be further increase to 68°C. These two parameters are known to enhance the stringency of hybridization.

i) K. brevis and K. mikimotoi probes will be tested with restriction-digested genomic DNA. The probes will be challenged with other non-related DNA Detection limits of ligation will be conducted with genomic DNA.

ii) Lineal and geometric rolling circle amplification will be developed with synthetic exonuclease treated products and genomic DNA. Optimization of RCA conditions will be determined at different incubation times and ligated product concentrations. The optimum conditions will be determined by the amount of generated products, which will be verified by agarose gel electrophoresis and quantified by spectrophotometry.

Dissemination Objectives for next reporting period
Funding was not allocated for the dissemination of results in phase I.

Overall Project Timeline Update
Second phase/First year ­Ligation specificity conditions will be optimized and tested with genomic DNA. K mikimotoi probe will be tested and validated. Detection limits of ligation conditions will be determined with genomic DNA and compared to those with synthetic targets. Development of assay conditions for rolling circle amplification.

2nd year. Optimization of the coupled PLP/RCA/Luminex system. Determine detection limits of the integrated system. Determine which amplification strategy works best, lineal or geometrical. Optimize DNA extraction methods from environmental samples. Collect environmental samples pre-, during, and post- K. brevis bloom. Run the assay on field samples and compare to microscopic enumeration.

Expenditures
The money allocated for the expenditures seems to be appropriated.

End User Advisor Feedback
Advisor: Dr. James Jacobson
Organization: Luminex Co.
Location: Austin, Texas
Phone number:512-219-8020
E-mail:jwjacobs@luminexcorp.com

At this stage, what are the potential applications for this research? Please discuss how you and others could potentially use the technology.
As elaborated by the researchers in this report as well as in the original proposal, harmful algal bloom (HAB) has significant economic and health impacts. Moreover, the extension can readily be made to other environmental monitoring situations where pathogens, contaminants, toxins, etc. decrease the economic value and / or safety to those using the beaches, fields, parks or similar areas. An additional connection can be made to monitoring for biowarfare agents.

Although the results are still early, progress to date is promising. A method that provides for rapid, simplified and distributed testing for these targets will have significant value across broad application areas including the project target ­ monitoring HAB. Rapid, localized testing for HAB, in particular, can lead to more efficient monitoring, predictability and response to the threat. The assay technology in development is consistent with our technology development plans to produce less expensive, ruggedized and portable analyzers.

What are the key challenges to application of this technology? Please consider the technology itself as well as issues related to regulation, politics, socio-economic pressures, trends in the field etc.
As the investigators point out, the project is still in early phases and much remains to be done in optimizing various parts of the analytical methods. That said, it is critical that the process ­ from sample collection through result reporting ­ be as simple and straightforward as possible. To provide for distributed testing, not only must the technology be relatively inexpensive, but the operator requirements must match the process. Many outstanding analytical methods have failed to find commercial success because they require highly-skilled (and thus highly compensated) operators. “Keep it simple” should be an ever present mantra.

Other concerns that must be addressed include the freedom to operate (FTO) with the proposed probe and target sequences, methods (e.g., LNA probes) and regulatory requirements. I am most familiar with the FDA-regulated environment so cannot offer much advice on the environmental monitoring arena but I suspect that relevant USDA and/or EPA requlations exist and should be contemplated in decisions about assay materials, result reporting and instrumentation registration, etc.

Has anything changed about this project's potential applicability since the last reporting period (not applicable to the first Progress Report)?
Questions/comments/ suggestions for the researchers?
I’ll just take this opportunity to congratulate the researchers on their early progress with this exciting project. Much remains to be done but the results to date are promising.