Part:BBa_K5087006
KacF
Introduction
The KacF primer is a part designed specifically for PCR amplification of a fragment of the Prymnesium parvum genome consisting of the ITS2 sequence.
Biology & Usage
Our team utilized this primer to obtain the fragment containing the ITS2 sequence of Prymnesium parvum, measure the DNA concentration, and subsequently use it in SHERLOCK assays. This was done to establish the limit of detection for both our plate fluorescence readout test and the PrymFlow lateral flow assay.
The ITS2 (Internal Transcribed Spacer 2) region is a non-coding segment of DNA found within the ribosomal RNA (rRNA) gene cluster. In the genome of Prymnesium parvum, the ITS2 region lies between the 5.8S and nuclear large rRNA genes [1].
The ITS regions, including ITS2, are commonly used for species identification because they tend to vary between species. This variability makes the ITS2 region an effective target for designing species-specific primers, such as those used to identify Prymnesium parvum [2].
Amplification of DNA
To make our test suitable for in-field use, we used the RPA (Recombinase Polymerase Amplification) method for DNA amplification. RPA is an isothermal amplification technique that works at a constant temperature [3], unlike PCR which requires thermal cycling. However, RPA presents challenges, particularly with DNA cleanup and concentration measurement after amplification.
Since RPA does not facilitate easy cleanup [4] or accurate DNA concentration measurement, we needed a method to ensure the precise amount of Prymnesium parvum DNA for our assays, especially those involving limit-of-detection testing. To address this, we initially performed a PCR step using KacF and KacR primers to amplify the DNA fragment. After amplification, we purified the DNA product, measured its concentration, and then proceeded with the RPA and SHERLOCK assays. This approach allowed us to obtain accurate measurements and reliable results for our tests.
The SHERLOCK method
The SHERLOCK [5] platform is a modern synthetic biology tool that utilizes the properties of the Cas13a protein, an enzyme from the Nobel Prize-winning CRISPR-Cas system. The Cas13a protein is guided with high specificity to the target sequence using crRNA.
The crRNA molecule is crucial for the assay's specificity. It consists of a direct repeat (DR) sequence and a spacer sequence that is complementary to the target. The crRNA molecule is designed to uniquely identify the organism by targeting the Internal Transcribed Spacer (ITS) sequence in its genome.
First, the Cas13a protein binds to the organism's genetic material, which was previously amplified using RPA and transcribed into RNA. Once bound, the Cas13a protein is activated and exhibits a "collateral" RNase activity, meaning it non-specifically cleaves nearby single-stranded RNA molecules [5].
This activity can be used in assays by including synthetic RNA probes tagged with a fluorescent reporter and a quencher in the reaction mixture. A fluorescent signal indicates that the reporters have been cleaved by Cas13, confirming the presence of the DNA target in the sample. The SHERLOCK method can also be used with Lateral Flow Assays (LFA) [5].
Experimental Validation
Purpose
The purpose of this experiment was to evaluate the usefulness of KacF and KacR primers for PCR amplification of Prymnesium gDNA, intended for use as input in future LFA (Lateral Flow Assay) tests.
This was done to address previously encountered issues with the low concentration of Prymnesium gDNA. By PCR-amplifying the detected fragment, we aimed to obtain a higher concentration of the target DNA for more reliable testing and to enable precise quantification of its concentration.
Direct measurement of gDNA concentration is unreliable because our algal cultures were established from an environmental sample taken from a water body affected by Prymnesium parvum blooms, meaning they are not pure cultures and may contain DNA from various other organisms. Consequently, the gDNA isolated from these cultures likely includes DNA from these other organisms in addition to Prymnesium parvum. When DNA concentration is measured using a NanoDrop Microspectrophotometer, it quantifies all DNA present in the sample, of which Prymnesium parvum DNA might constitute only a small percentage.
Methods
Preparation of PCR Reaction:
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We mixed the following components in a PCR tube, adjusting the volume as needed for our experiment.
The melting temperature (Tm) of the primers was calculated to be 70°C using the NEB Tm calculator.
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We gently mixed the reaction components and, if necessary, collected all liquid at the bottom of the tube by a quick spin.
Thermocycling Conditions:
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We transferred the PCR tubes to a PCR machine with a heated lid, preheated to 98°C.
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We set the thermocycler with the following parameters:
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Pre-denaturation: 98°C for 30 seconds
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Denaturation: 98°C for 10 seconds
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Annealing: 70°C for 30 seconds
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Elongation: 72°C for 1 minute
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Final extension: 72°C for 2 minutes
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Number of cycles: 40
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Electrophoresis:
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We prepared a 1% agarose gel.
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For each PCR reaction, we mixed 5 μl of the PCR product with 1 μl of loading dye, achieving a final volume of 6 μl.
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We loaded the samples onto the gel, along with 5 μl of a DNA ladder (1 kb Plus DNA Ladder, New England Biolabs) on both sides of the gel.
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We carried out electrophoresis for 35 minutes at 90 V.
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We visualized the DNA bands under UV light. The target Prymnesium parvum product was expected to be 672 bp in length.
Results
Figure 2: PCR results using KacF and KacR primers. Well 1: Ladder, Well 2: Negative control, Wells 3 and 4: Samples containing Prymnesium parvum DNA.
Conclusions:
A product of the correct length (672 bp) has been obtained, which can be used for future experiments. The primer set works reliably in the conditions optimized by our team.
Parts Compatible with KacF
This part should be paired with the KacR (BBa_5087007) primer for PCR reactions.
Sequence
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 7
- 1000COMPATIBLE WITH RFC[1000]
Primer and crRNA Collection Binding Sites
Here, we illustrate the positioning of all primers and crRNAs in our PrymDetect Toolkit on the ribosomal cistron of Prymnesium parvum genomic DNA.
Biosafety
We used the Asimov's tool — Kernel — to check the sequence's safety with the Biosecurity Sequence Scanner. The results showed no flagged sequences, confirming that this part is safe to use.
Resources
- [1] White, T.J., Bruns, T., Lee, S., & Taylor, J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: A Guide to Methods and Applications Academic Press. Published online 1990.
- [2] Galluzzi L, Bertozzini E, Penna A, et al. Detection and quantification of Prymnesium parvum (Haptophyceae) by real-time PCR. Lett Appl Microbiol. 2008;46(2):261-266. doi:10.1111/j.1472-765X.2007.02294.x
- [3] Lobato IM, O’Sullivan CK. Recombinase polymerase amplification: Basics, applications and recent advances. Trends Analyt Chem. 2018;98:19-35. doi:10.1016/j.trac.2017.10.015
- [4] TwistDx Ltd. (2023). TA01C user manual (Rev. 1, Version 2). TwistDx. Retrieved from https://www.twistdx.co.uk/wp-content/uploads/2023/12/ta01cmanual-combined-manual_rev1_v2.pdf
- [5] Kellner, Max J., Jeremy G. Koob, Jonathan S. Gootenberg, Omar O. Abudayyeh, and Feng Zhang. “SHERLOCK: Nucleic Acid Detection with CRISPR Nucleases.” Nature Protocols 14, no. 10 (October 2019): 2986–3012. https://doi.org/10.1038/s41596-019-0210-2
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