Team:Cork Ireland/Project
Summary
Our project is a novel bacterial method of DNA detection. We have developed a customisable, linearised, double stranded plasmid with two sticky overhangs. When the sticky overhangs come into contact with a target sequence, the binding of the DNA sequence to the overhangs circularises the plasmid. The circularised plasmid is then transformed into competent E. coli cells. Bacterial growth of green fluorescent colonies indicates a positive result, therefore the complementary DNA target sequence was present. This system could act as a cheap alternative to both digital and real time PCR, as target DNA fragments are amplified in living cells without the use of a costly PCR machine. This system could potentially be used as a diagnostic or screening tool for viral and/or bacterial infection such as Human Papilloma Virus, Mycobacterium tuberculosis. By improving sensitivity and specificity this system could also be used for the detection of genetic mutations resulting in disease such as cystic fibrosis.
Over the summer we have:
- Standardized the readout of the Basehunter system.
- Developed a new method for detector plasmid preparation with the goal of reducing false positive results improving sensitivity
- Characterized the specificity of the Basehunter system
- Developed detectors for shorter target sequences to improve specificity
- Designed a new mycobacterium detector based on community consultation
- Created a prototype of the Basehunter system that was shipped to and successfully used by a collaborating iGEM team.
Our Project
Basehunter is a bacterial-based DNA detection system. This project aims to further develop the specificity, sensitivity and colour readout of the system. Building on last years work, we have designed customizable plasmids that could be modified to detect any specific short DNA sequence. Bacterial growth is a readout for the presence of target DNA.This system potentially provides a cheap, simple alternative to both digital and real time PCR as target DNA fragments are amplified in living cells. Thus making this system applicable in poorly resourced labs in both the developed and developing world as PCR machines are expensive to purchase and can be costly to run.
The system works by interaction of the target sequence with the plasmid, and upon subsequent transformation into competent E.coli, results in bacterial growth and the production of a fluorescent signal. The plasmid is linearised with overhanging sequences that are complementary to the target sequence. Upon annealing to the target, the plasmid becomes circularised and functional in a bacterial cell. The presence of this fluorescent signal indicates that the target DNA is present. In this project we have been investigating different possible methods of detector (plasmid) construction. The sensitivity of the different detector types has been tested thoroughly. We have also been improving the colour readout of the system.
Three different types of DNA detector have been constructed; SRY detector, that detects a DNA sequence only present on the Y chromosome. This is used to investigate the systems ability of detecting target sequences in genomic samples. A HPV detector that detects a sequence in the L1 gene that encodes the capsid of the Human Papilloma Virus 16 & 18. We are investigating different target and detector lengths to see which is both the most sensitive and specific. Lastly, we have also made a detector for Mycobacterium tuberculosis.
The specificity of these detectors has been studied by different detector reactions using mutated targets. Increasing the specificity is important as in order to be used as a diagnostic test, the Basehunter system must have a high specificity to avoid a false positive result.
The colour readout of the system allows us to identify if the target DNA was present or not. Two different fluorescent colours are present, one acting as a positive readout if the DNA target is present. Another colour acts as a transformation efficiency control. The colour readout was altered by producing different functional fluorescent proteins by a series of ligations. These were then tested and used, thereby significantly improving the colour readout of the system.
Cork iGEM aim to further optimise the Basehunter system to detect other DNA sequences from bacterial or viral pathogens, such as Mycobacterium tuberculosis, and genetic mutations that cause diseases such as cystic fibrosis. We also hope to continue to test the sensitivity and specificity of the detector plasmids in order to optimise the system and render it suitable as a diagnostic test. Other potential applications may include using the system to estimate copy number variation in oncogene amplification and also in multiplex detection to detect a number of targets at one time.
In our development of this diagnostic system, we have consulted with expert biomedical scientists in both the developed and developing world to discuss the specificity and sensitivity of the test. We also got advice on what diseases lack a simple diagnostic test, to see where our project could be implemented.
In addition to this we have investigated the role iGEM has had in emerging synthetic biology start up companies through interviews and surveys conducted with companies taking part in this years IndieBio Start up Accelerator Programme.
Basehunter - aims to create a system for bacterial-based DNA detection. Last year, the UCC Ireland 2014 Team designed customizable plasmids that can be activated to detect any specific short DNA sequence. This system potentially provides digital quantification of target DNAs – representing an alternative to digital and real-time PCR. Interaction of the target sequence with the plasmid and subsequent transformation into E. coli results in bacterial cell growth and production of a visible fluorescent signal. As proof-of-principle they also designed a HPV-detector plasmid that detects a short sequence from the human papilloma virus. This could be a rapid and cheap diagnostic tool for pathogenic DNA, and a revolution in resource poor hospital labs in developing countries or in an agricultural or industrial setting.
Standardization of Basehunter Readout
Introduction to Results
Standardization of Basehunter readout
Summary:
Basehunter relies on transformation of detector/target complexes into E. coli with subsequent colony growth as the readout. The efficiency of bacterial transformation is thus a key variable in the Basehunter protocol. We developed a method to have a built-in control for transformation efficiency in every Basehunter detector reaction as outlined below. This allows results to be reported in standard units that we refer to as Controlled Colony Count (CCC):
The problem:
Transformation efficiency varied greatly between different batches of cells, different experiments and even within experiments. As seen in figure 1, on occasion CFUs achieved did not correlate with concentration of plasmid transformed even within the same experiment. Clearly variation in transformation efficiency will have a significant impact on results obtained with Basehunter detectors and must be controlled for.
The solution:
We reasoned that a plasmid that produces colonies that are distinguishable from those from our detector reaction could be used as a built in transformation efficiency control. Thus detectors were generated from plasmids encoding constitutively expressed GFP and used in combination with a known amount of a control (uncut) plasmid encoding RFP. GFP+ and RFP+ colonies could be distinguished by eye, or with the aid of a blue LED lightbox. In some cases, a basehunter detector without a fluorescent marker was used in combination with a GFP expressing control plasmid, but the principle is the same (e.g. SRY detector). The results of simultaneous transformation of the detector and control plasmid may be seen in figure 2.
Since a known amount of the control plasmid is simultaneously transformed with the detector we can calculate the transformation efficiency of each test and express the results in standard units that that acccount of variations in transformation efficiencies. The calculated transformation efficiency is used to amend the number of detector CFUs achieved to a constant transformation efficiency (10^6 CFU per ug DNA). We refer to this as the Controlled Colony Count (CCC). Hence results of all experiments could be compared. Calculations for all experimental results were performed as outlined in Table 1 and then plotted as bar graphs.
Sample calculation of the Controlled Colony Count:
- A 1:1000 dilution of control plasmid was used of concentration 114ng/ul, ie. 0.114ng/ul.
- 1 ul of this dilution was added to the transformation, meaning 0.114ng were transformed and all of this was plated.
- Average control derived CFU achieved was 1429.33
- Transformation Efficiency (TE) = (colonies on plate) / ng of DNA plated x 1000ng/µg (iGEM Competent Cell Test Kit Protocol, 2015)
- TE = 1429.33/0.114 X 1000ng
- TE = 1.2 x 10^6
Selection & Design of Basehunter Detectors
In the course of this project we used detectors that are designed to detect three different targets:
- Human Papilloma Virus (HPV) (K1698001)
- The Sry gene located on the Y chromosome (K1698002) & (K1698003)
- Mycobacterium species (K1698004)
These targets represent the major applications that we forsee for Basehunter – namely the detection on infectious agents including (viruses and bacteria) and the diagnosis of genetic conditions (inherited mutations, copy number variations etc.). Further information about each these targets is provided below.
HPV Detector
Human Papilloma virus
HPV is a common pathogen worldwide. Moloecular diagnostics has helped saved over 275,000 lives every year and our team believes our device could be of use in this setting. The human papilloma virus (HPV) is a small double-stranded DNA virus with a genome of 8000 base pairs. It infects the epithelial cells of skin and mucosa (Cutts et al., 2007). Theses epithelial surfaces (squamous cells) include all areas covered by skin and/or mucosa such as the mouth interior, throat, tongue, tonsils, vagina, cervix, vulva, penis (the urethra - the opening), and anus. Transmission of the virus occurs when these areas come into contact with a virus, allowing it to transfer between epithelial cells. Over 170 HPV types have been identified and, each HPV strain is denoted by a number. Of all the HPV variants, HPV16 is one of the most carcinogenic "high-risk" HPV stains. HPV16 is implicated in oropharyngeal (throat) cancer, while it also plays a role in cervical cancer (Muñoz et al., 2003).
It has been postulated that HPV16 infects epithelial tissues through micro-abrasions of epitheliums; here HPV16 partners with receptors located on the basement membrane such as alpha integrins and laminins. This leads to entry of the virus into basement membrane cells. HPV16 accomplishes entry via clathrin- and caveolin-mediated endocytosis (Laniosz et al., 2009) . At this point, the viral genome is transported to the nucleus where it establishes itself at a copy number between 10-200 viral genomes per cell. HPV Type 16, causes the most incidences of cervical cancer cases worldwide, hence a target from this genome (Ref: NC_001526) was chosen.
HPV16’S L1 gene codes for the L1 protein, the major capsid protein of HPV16. By spontaneous self-assembly, five of these L1 proteins form a homopentamer called a capsomere (Heino et al., 1995).72 of these capsomeres come together to form a capsids. The capsomeres are held together by disulphide bonds and the minor capsid protein L2, which stabilise the overall capsid structure.
A 55bp target sequence that is released from the L1 gene by digestion with HaeIII restriction enzyme was chosen as the basis for a HPV Detector (Figure 1). Some of us working as part of a secondary project of the 2014, UCC iGEM team demonstrated that this HPV Detector was capable of detecting a single stranded 55bp Target as well as a double stranded 55bp target. A double stranded target could also be detected in a context of where other HaeIII DNA fragments was present in the form of fragments of a HaeIII digested plasmid indicating a degree of specificity for the correct target sequence. This “55bp HPV detector” has been further studied and optimized in this years project in order to submit a well characterised Biobrick Part to the Registry (BBa_K1698001). (No detector parts were submitted in 2014)
References
- Cutts, F. T., Franceschi, S., Goldie, S., Castellsague, X., De Sanjose, S., Garnett, G., Edmunds, W. J., Claeys, P., Goldenthal, K. L., Harper, D. M. & Markowitz, L. 2007. Human papillomavirus and HPV vaccines: a review. Bull World Health Organ, 85, 719-26.
- Heino, P., Dillner, J. & Schwartz, S. 1995. Human papillomavirus type 16 capsid proteins produced from recombinant Semliki Forest virus assemble into virus-like particles. Virology, 214, 349-59.
- Laniosz, V., Dabydeen, S. A., Havens, M. A. & Meneses, P. I. 2009. Human papillomavirus type 16 infection of human keratinocytes requires clathrin and caveolin-1 and is brefeldin a sensitive.J Virol, 83, 8221-32.
- Muñoz, N., Bosch, F. X., De Sanjosé, S., Herrero, R., Castellsagué, X., Shah, K. V., Snijders, P. J. F. & Meijer, C. J. L. M. 2003. Epidemiologic Classification of Human Papillomavirus Types Associated with Cervical Cancer. New England Journal of Medicine, 348, 518-527.
SRY Detectors
Sry is a gene located on the Y chromosome. It codes for sex-determining region Y (SRY) protein which is also known as Testis-determining factor (TDF) (Kashimada and Koopman, 2010). SRY protein is transcription factor that is responsible for male sex determination in mammals.
A target located on the Y chromosome was chosen to test whether our Basehunter detector could be used for real world applications because it would allow us to test both the sensitivity and specificity of the system in detecting a target sequence from genomic DNA. The ability to detect and quantify target sequences from a genomic DNA sample would provide evidence that the Basehunter detector could be used for genetic testing of either inherited diseases for example. By choosing a target located on the Y chromosome, it will be possible to test the detector with genomic DNA from a male and to use genomic DNA from a female as a negative control. This would be a very good test of the sensitivity and specificity of the Basehunter detection system.
Two Sry detectors were used in the project:
- A detector specific for a 62bp target that is generated from the Sry upon HaeIII digestion was developed by some of us working as part of a secondary project of the 2014, UCC iGEM. This detector had already been shown to work in principle but has been further characterised this year and submitted to the Registry as Part Number (BBa_K1698002).
- A second Sry detector was also developed that recognises a 32bp PstI fragment from the Sry gene has also been developed Part Number (BBa_K1698003).
References
- Kashimada, K., Koopman, P. (2010). Sry: the master switch in mammalian sex determination.Development. 2010 Dec;137(23):3921-30
Mycobacterium detector
From our policy and practice work during the summer we learned of the growing need for a detector to quickly analyse whether or not a patient was infected with a mycobacterium like M.Tuberculosis which is still a major cause of death in many third world countries. TB is second to aids/HIV as the most deadly disease caused by an infectious pathogen. We have known of the existence of the pathogen behind TB since the beginning of the 20th century , however to this day every person with active TB will go on to infect 10 to 15 more people ( World health organisation 2010 ). Mycobacterial culture on solid Lowenstein-Jensen (LJ) medium is considered the gold standard isolation method (Conde et al 2009). Although the limitation of this method is the long incubation period (2-8 weeks), it is used by most developing countries because of its low cost. Techniques such as nucleic acid amplification and automated liquid culture systems are costly and depend on sophisticated tools, which prevents their routine use in poor countries. TB is diagnosed currently using acid fast stains which is based on the binding of mycobacterium like M.Tuberculosis to fuchsin which is very selective. Our detector serves as a novel way to amplify DNA in ecoli and the results of the amplification are evident in the number of colonies that form on the plate.In saying that statistics show that over 37 million lives are saved every year due to effective diagnosis. We constructed a detector which would act as a negative screening device for TB. We decided to this based on the reported growing need which we identified in our policies and practices throughout the summer. However proper treatment is available and we decided to construct our detector to allow for a cheap diagnosis of the M. Tuberculosis pathogen.
Target Identification
Mycobacterium are unique among bacteria in that they possess several unique proteins involved in fatty acid synthesis and metabolism. By identifying such proteins which are unique to mycobacterium tuberculosis (Beile Gao et al 2006), we were able to obtain the gene sequence for the protein and establish a detector target from this sequence. One protein which contained a 24 base pair target flanked by Sma1 restrictions sites was identified, ML0319 is a lipoprotein uniquely found in Mycobacteria and also present in Norcardia. The detector construct designed to detect this target is shown in the figure below. The nucleotide base sequence for this gene has already been sequence and we hoped to be able to diagnose TB using our detector. Due to the high GC nature of the 24 base pair target sequence, we identified that BamHI and KpnI would be the restriction sites to be used in the actual detector. The proposed target nucleotide was checked to see if it would have a likely chance to form hairpin structures or if we would experience any problems with annealing or dimerization. Even though the target has a high GC content ( 68 %), it will likely to not form hairpin structures spontaneously.
Construction and Testing the Mycobacterium detector
The Mycobacterium detector was constructed after the Mycobacterium detector sequence was cloned into the pSB1-C3 containing the GFP generating biobrick part K584001. It was also assembled by cloning straight into the digested linearized plasmid pSB1C3, oligonucleotides were ordered from IDT and they were first phosphorylated and then annealed together (SEE PROTOCOL SECTION). This annealed detector sequence was then ligated into the digested linearized plasmid backbone and transformed. A four enzyme digest was done i.e the enzymes Nt.BspQI, Nb.BtsI, BamHI and KpnI were used as described in the protocols section. Following the digest, the mixture was ran and a gel and a gel extraction was done to remove the top strand i.e detector plasmid, which could then be used in the detector reaction.
Results
References
- World Health Organization. Global tuberculosis control report 2010. Geneva: World Heath Organization; 2010.
- Conde MB, Melo FA, Marques AM, Cardoso NC, Pinheiro VG, Dalcin Pde T et al. III Brazilian Thoracic Association Guidelines on tuberculosis. J Bras Pneumol. 2009;35(10):1018-48.
- Gao B, Paramanathan R, Gupta RS. Signature proteins that are distinctive characteristics of Actinobacteria and their subgroups. Antonie Van Leeuwenhoek. 2006 Jul;90(1):69-91. Epub 2006 May 3. PubMed PMID: 16670965.
Results
Part One
Optimising Basehunter Sensitivity
A. Alternative Detector Construction by PCR to reduce detector background
Summary:
The initial method for detector preparation by digestion of plasmid containing the detector part with restriction and nicking enzymes was found to give a low level of background colonies, presumably due to incomplete digestion of the plasmid. Such false positives are not acceptable in a diagnostic test. Also the level of such false positives will increase if higher transformations efficiencies are employed to boost the sensitivity of the system. We therefore devised a method to amplify the detector by PCR. Preparation of the the activated detector by dgestion of linear PCR products largely eliminated background colonies.
UCC iGEM 2014 developed the first method of detector construction by digestion using 4 enzymes (Kpn I, HindIII & nicking enzymes, NtBsI & NtBsQI) (click here to read our protocols). Results of this method of construction were generally positive as positive control plates achieved colonies TNTC and negative control plates showed <10 CFUs. As seen in figure 1 and 2, showing detector reactions using digested detector made by UCC iGEM 2014, tested in June 2015 & September 2014, negative plates showed CFUs. This may have been due to the presence of undigested detector that transformed without target needing to be present. To improve on this, Cork iGEM 2015, created a new protocol for detector construction by PCR.
The new protocol (click here to read our protocols) involves using primers specific to the detector part and amplifying this part on a plasmid containing GFP biobrick and chloramphenicol resistance. These primers are illustrated in Figure 2. Further modification of the detector is then carried out to activate.
A DpnI digest after PCR ensures that template DNA is digested, being rich in GATC restriction sites. A subsequent clean up step ensures that template DNA is removed to minimise re-annealing to detector. Next, an enzyme digest was carried out using the same 4 enzymes as the original protocol (NtBsQI, NtBsI, KpnI & HindIII), shown in Figure 2. This was done to create single stranded overhangs complementary to the target. The detector was then reacted with decoy oligos (complementary to the top strand of the detector, which is to be removed) in excess to yield the single strands of the detector. Another cleanup was then carried out to remove the decoys. Further experimentation showed that the decoy oligo step was unnecessary and the detector was ready for use after the 4 enzyme digest and PCR cleanup.
Table 1 - Results of detector reactions using the PCR constructed detector showed less background overall. However, the number of CFUs achieved on positive control plates also showed a reduction. This may have been due to DNA concentration in the final detector sample being less after multiple clean ups during construction. This may be seen in Table 1 which shows a comparison of detector reactions carried out simultaneously using digested and PCR constructed detectors.
B. Detection of Target in Genomic DNA
Our gold standard for the sensitivity and specificity of the Base hunter system would be that it should be able to detect a sequence from a sample of restriction digested genomic DNA. Here we employed the SRY detector, specific to the SRY gene on the Y chromosome. Digested female genomic DNA serves as a very stringent negative control. Our results show some signs that the Sry target is being detected, but also that non-specific interactions are yielding positive colonies from female DNA.
Sry 55bp Detector
The Sry 55bp detector was tested with single stranded, double stranded and double stranded targets with background DNA present in 2014 but the detector had yet to be reacted with genomic DNA. This was initially carried out by Srijita Banarjee as a final year research project, and again repeated by Cork iGEM 2015 using the detector constructed by the new PCR amplification protocol (click here to read our protocols).
As shown in figures 2 the digested detector appeared to yield more colonies with male genomic DNA, however the oppositie result was seen in figure 3. Results appeared inconclusive and it emerged that the detector faces a major challenge in use with samples with a lot of background DNA, such as a patient genomic sample. Sequencing of colonies from these experiments however showed that colonies from the male genomic DNA had correctly picked up the Sry target, while does from the female genomic DNA had deletions in the region of the detector. A better understanding of the mechanisms of how non-specific interactions from female DNA results in plasmid recircularization may allow this background to be eliminated.
Sry 32bp detector
To improve on this result, a smaller SRY detector was constructed (32bp). A smaller detector may have been more specific to its target and hence give less false positives. Also, the 32bp target site was nested in 2 PstI restriction sites in the genome. This meant that digestion of a genomic sample with PstI yielded fewer fragments as a 6 cutter enzyme, than HaeIII which was previously used. Unfortunately due to time constraints the SRY 32bp Detector was not tested with genomic DNA.
Part Two
Assessment & Optimisation of Specificity
A. Design of Detectors for Shorter Targets
Summary
We reasoned that detectors design to recognize shorter targets might exhibit more sequence specificity. We therefore tested variants of the HPV detector as well as a new Sry detector designed to detect target with a size of 30-32bp. The performance of these detectors was similar to the longer 55 and 62 bp detectors that we orginally designed. Thus Basehunter can work for target sequences in this shorter size range.
Development of detectors for shorter target sequences:
In 2014, UCC iGEM demonstrated that the HPV detector was capable of detecting a single stranded 55bp Target, a double stranded 55bp target and a double stranded target where background DNA was present in the form of fragments of a digested plasmid.
Our aim this year was to further test this detector in order to try and optimise the system. This involved changing the target length and creating several HPV detectors and targets. Our thoughts were that a smaller “non-target” or non-complementary nucleotide would have a smaller chance of binding unspecifically even if some of the bases were complementary in that there would be a smaller amount of hybridisation between the two DNA strands i.e detector and target. We ordered primers so that we could amplify the detector by PCR to create detectors that would bind to targets of different lengths e.g. complementary oligonucleotides.
30bp and 24 bp variants of the HPV detector were also constructed and assessed to see if they were more selective than a larger detector. Results shown in figure 1 comparing the HPV 30 bp and 55 bp detectors suggest that the 55bp Detector gave slightly more positive colonies and somewhat fewer colonies on negative control plates. Thus the 55 bp detector size worked better somwhat better. Nevertheless the 30bp detector did work quite well and number of colonies on the control plates made be more related to variation in the preparation of the detector (incomplete digestion) rather than length of the target per se. Further testing could be carried out to determine the exact optimal length of a detector region.
Assembly of a new Sry detector
Giving our thoughts that a shorter detector might be more useful, we constructed a 32 bp Sry detector. This detector was PCR amplified using primers ordered from IDT and the original template of the 62 bp Sry detector. This detector was tested using genomic DNA digested with Pst1 since digest with the six cutter will result in fewer smaller DNA fragments being made that might hybridise on unspecifically when compared to the target being made after the genomic DNA was digested with HaeIII.
Studies were done to investigate digested genomic DNA as a target for the detector using both the Sry 62 bp detector and the Sry 32 bp detector. The Sry detector was tested and checked to see if it worked using a positive target that was ordered from IDT. The results show that the detector that was created worked.
To evaluate the reliability of the detector to select the target of the correct length, targets of various size (with deletions in true target sequence) were reacted with the detector. First tested was the 30bp Detector with targets ranging in size from 18bp to 30bp, with missing sequence at ends of 30bp target. Figure 1 shows that the detector reacted significantly more with the target of the correct length than those of smaller length, in spite of complementary sequence to the middle region of the detector.
Figure 2 shows the effect of targets longer than 30bp on the detector reaction.
Results suggest that a longer target, although giving fewer CFU than an accurately sized target, gives more CFU than a target that is too short (see Figure 1). In addition, random sequences which are either accurately sized or too short are not detected by the detector (Figure 3).
C. Testing Detector with Mutated Targets
An important aspect of a reliable diagnostic test is its ability to differentiate the true target and other components in the sample.
This eliminates false positives and is referred to as “Specificity”. To assess the system’s specificity, it was reacted with a number of targets
which resembled the true target in size and sequence. The mutated targets varied either by a deletion, insertion or the presense of
incorrect bases in part or all of the target (shown in figure 1).
The 55bp Detector was first tested with the mutated targets. Results are as shown in figure 2.
Figure 2 suggests that more colonies are achieved when the correct, non-mutated target is present (Wild Type (WT)). However, CFUs were still seen where mutated targets were present. This was especially true where the mutation occurred at the ends of the target (deletions @bases 1-3, insertions at base 55). Where a mutation occurred in the middle region of the target (base change at 26-28bp) no colonies were seen, suggesting the middle region of the detector is highly specific, while the detector is inefficient at detecting targets with mutated ends.
Part Three
Further Characterisation
A. Assessing need for Phosphorylation of Detectors & Target
We also carried out experimentation using the Sry detector to try and understand some of the mechanistic aspects of the detector. One of the experiments that we carried out was to see whether the detector reaction results would be impacted if non-phosphorylated DNA was used as target and detector, we hypothesized that doing the detector reaction with phosphorylated target and detector should give more CFUs than if both non-phosphorylated target and detector were used.
As shown by the data there wasn’t quite a big difference in corrected colony count between the three tests and the positive. We hypothesized therefore that DNA polymerase I in E.coli might be chewing back on the non-phosphorylated DNA because of its exonuclease activity in recognizing a possible error and the polymerase actively filling in nucleotides that are phosphorylated for ligase activity (Khare & Eckert, 2002 ). This could be probable since the DH5 alpha cells used in our lab don’t have polymerase I knocked out. The results show that the three tests on average do not differ significantly from the positive control.
References
Khare V, Eckert KA. The proofreading 3'-->5' exonuclease activity of DNA polymerases: a kinetic barrier to translesion DNA synthesis. Mutat Res. 2002 Dec 29;510(1-2):45-54. Review. PubMed PMID: 12459442.
B. Genomic DNA- unspecific digested targets
Genomic DNA
We also tested the selectivity of the detector system using the three HPV detectors (see selectivity section). Experiments were trialled where digested genomic DNA was used as a target, any small cut pieces of DNA were hypothesized to be unspecific.
We digested the genomic DNA with the four cutter Haelll and then carried out the detector reaction using this digested DNA. We hypothesized that HaeIII would cut frequently enough throughout the genome so that nucleotides of appropriate length would be available to anneal on if the detector system was unspecific. The results show that the detector was specific enough so that there was very little background.
This experiment was repeated using the HPV 30 base pair detector using high efficiency cells to test whether or not there would be no background still. High efficiency cells were commercially bought from NEB.
This experiment was repeated using the HPV 30 base pair detector using high efficiency cells to test whether or not there would be no background still. High efficiency cells were commercially bought from NEB.
Conclusion
The detectors constructed work efficiently to differentiate samples containing target sequence and those without DNA. Where background DNA is present in the sample, such as genomic samples, the detectors work less specifically and can return false positives. Efforts to improve this, such as constructing shorter detectors and using enzymes that cut less frequently to digest genomic samples were began but results were unclear at the time of submitting project. Further work may be done to improve this.
Also noted was the ability of the detector to accurately select targets which were longer than the detector itself but not those which were shorter. This feature may allow the detector to be constructed shorter than the intended target sequence. This is favourable as shorter detectors tended to perform better and return higher CFU counts.
The customizable-aspect of the detector design appears to hold true as detectors for mycobacterium species and different sized targets for HPV and SRY gene were quickly and easily constructed. In a matter of days, a unique detector can be designed and constructed at minimal cost for virtually any target. These detectors appear to work equally well. This suggests that the detector holds promise as a diagnostic tool applicable to any DNA-detecting purpose.
Interpretation of results was improved significantly this year as the standardisation to a specific transformation efficiency and use of a control RFP plasmid in all tests ensures reliability of results and the ability to compare with those obtained at different times or in different labs.