Project 4X200 – Real-Time Detection of DNA
The goal of Project 4X200 is to quickly identify specific DNA sequences from small samples of biological fluids like blood or saliva. The aim is to detect DNA from 200 different agents using a drop of these fluids within 200 seconds. This rapid testing could be vital for quickly identifying biological threats at places such as airports or other entry points.
This is a project idea I created with a proposal in the The Heilmeier Catechism, DARPA format.
Real-time detection of DNA for biosurveillance. A goal of detecting one of 200 agents with 200 nucleotides markers in 200 seconds or less. Samples of 200 microliters of a biological liquid (e.g., blood, saliva, etc.). The goal called 200,200,200,200 or 4X200.
Preparing Sample
Preparing the sample for DNA detection likely includes DNA extraction. Current DNA extraction methods take 10 minutes to 2 hours. A crude DNA extraction method needs to be developed to be done during other processes of DNA detection (Denaturation, Annealing).
Advantages and Challenges of Polymerase Chain Reaction
Polymerase Chain Reaction (PCR) is a widely used method for amplifying DNA, but its current implementation has several time bottlenecks that prevent it from being completed in 200 seconds. These bottlenecks include Denaturation (double strand DNA is separated into single strand DNA), Annealing (primer DNA used to start the DNA synthesis attaches to the single strand DNA), Extension ( DNA polymerase to synthesize the new DNA strand by adding nucleotides to the primers).
For sensing DNA, PCR is used as a test to determine if the primer DNA is present in the sample. If PCR was setup correctly and PCR did not occur, then it is assumed that the target DNA of the primer is not present. The advantage of PCR is that the sample DNA exponentially increases, making the target DNA easier to detect.
The disadvantage of PCR is time. A typical diagnostic PCR test takes 15-20 minutes. A test for biosecurity, screening a possible pathogen would need to be much faster than 20 minutes.
New Methods for DNA Detection
The most important step of PCR for DNA detection is the annealing of a primer with the target DNA with the sample DNA. For the PCR process extension is not needed. Denaturation is needed, assuming the sample DNA is double stranded. Denaturation takes 2-5 minutes and must be accelerated to meet the 200 second threshold of project 4X200.
Accelerated Denaturation – 60 seconds
The hydrogen bonds holding two DNA strands together are typically broken using heat (94-98°C).
Helicase
Helicases are enzymes that unwind DNA, adding these enzymes during this step could make the process faster.
DNA Binding Proteins
Proteins that bind to DNA such as Cold-shock proteins, which can bind and destabilize DNA, making denaturation occur at lower temperatures and might speed up the process.
Annealing – 30 seconds
The primer length could decrease the amount of time for annealing, however it would have the trade-off of the specificity and accuracy of the test.
Magnesium ions, polymerase, and other items could be added to increase the efficiency of annealing.
Detection – 20 seconds
With PCR the assumption is that if the PCR process occurred the target DNA (known primer) is in the sample DNA. For a simplified process like this the assumption would be if double stranded DNA is present, then the DNA bound with the target DNA (primer). Spectrometry is the measurement of the interactions between light and matter. Emitting and measuring different wavelengths of light the presence of double stranded DNA can be detected. The challenge using this method while not using PCR is the concentration and ratio of double stranded DNA. New spectrometry tools need to be developed to detect the presence of double stranded DNA at low concentrations.
Instead of spectrometry other sensor methods could detect bound DNA including: piezoelectric, magnetoresistive, electrochemical.
Unusual Ideas
In the original 4X200 project idea a few unusual ideas where purposed, including Cyborg Rats Sniffing DNA (Brain-to-machine interface on rats that are trained to sniffing the DNA strains), Genetically Engineered Cephalopod Skin (Skin Changes Color to DNA fragments). The idea for these ideas is based on detection.
Here are a few of my unusual ideas for project 4X200:
Cyborg Rats Sniffing DNA
This idea involves using brain-to-machine interfaces (BMIs) to enhance the natural olfactory abilities of rats for detecting specific DNA sequences. This concept leverages the highly sensitive sense of smell in rats, combined with advanced neurotechnology, to identify DNA strains quickly and efficiently. The detection of target DNA using olfactory senses with the quick data collection of BMI. Artificial intelligence could further extend this model by being trained on the BMI output. Training a rat to sense the target DNA, and training an AI model to determine the signal.
Genetically Engineered Cephalopod Skin
The concept of using genetically engineered cephalopod skin that changes color in response to DNA fragments represents an innovative approach to rapid DNA detection for the 4X200 project. By leveraging the natural capabilities of cephalopod skin and combining them with advanced genetic engineering, this method could provide a quick, intuitive, and scalable solution for detecting DNA. Cephalopod have chromatophores, sacks of pigment that they expand or contract to display different colors. This color-changing ability is used for camouflage and hunting.
One method to detect DNA would be to design DNA probes that can hybridize specifically to target DNA sequences. These probes are linked to a signal transduction mechanism that activates the color-changing proteins in the cephalopod skin.
G Protein Channel DNA Detection
The idea involves using G-protein-coupled receptors (GPCRs) that are engineered to bind specifically to target DNA sequences. Upon binding, the GPCR activates an ion channel, leading to a change in ionic current that can be measured as an electrical signal indicating the presence of the target DNA.
Adapting the Nanopore Sequencing Technology
Utilize nanopore sequencing, which involves passing DNA molecules through tiny pores and measuring changes in ionic current to identify specific sequences. Direct and rapid sequencing, potentially identifying DNA in real-time.
Surface-Enhanced Raman Spectroscopy (SERS)
Is a technique where molecules adsorbed on rough metal surfaces or by nanostructures, changing the properties of the surface, which can be detected with spectroscopy. For 4X200, SERS are applied to detect DNA by using Raman-active substrates that enhance the Raman scattering signal when DNA binds to them.
Quantum Dot-Based Detection
Use quantum dots conjugated with DNA probes that emit fluorescence upon binding to target DNA sequences.
CRISPR-Cas13-Based Detection
Utilize the CRISPR-Cas13 system, which can be programmed to bind specific RNA sequences. Spectrometry could then be used to detect if binding to the target DNA occurred.
