Many samples taken from individuals and animals are stored by treating them with chemical, formalin, and then encasing them in paraffin or wax;these are called formalin fixed paraffin encased (FFPE) samples. Usually this storage is very good for preserving them for observation under microscopes, but it presents some problems in terms of analysis of their DNA. Genetic analyses are important because many of these preserved samples are biopsies of cancer tissue, and cancers are diseases brought on by changes in the DNA. By understanding the genetic content of these samples, we can look for specific DNA sequences that can be used to diagnose cancer. We can also use the information in these samples to determine what therapies might work best, and to measure how well the therapies applied are working. There are some instruments and kits of reagents available for getting at the DNA in FFPE samples, but they are usually complicated. Often they use dangerous chemicals. They are inconsistent in the quality of DNA extracted, which can sometimes end up broken into small pieces or bound with proteins that make them unusable for genetic analysis techniques like sequencing, where the order of the different components of DNA are read out. Often they just extract too little of the DNA. Adaptive Focused Acoustics (AFA) is an ultrasonic technology that focuses sound energy onto small volumes. It is used for many processes like breaking up tissue samples to extract proteins and DNA and grinding solids into uniformly-sized particles. Recently AFA was applied to FFPE samples in a process performed manually. The process removes the paraffin and breaks the tissue down to extract the DNA. It provides 2-3 times as much DNA as other methods, and that DNA is of higher quality. This project will develop a system that uses AFA for FFPE samples with a special instrument and a special plastic cartridge. A vacuum pump on the instrument will move the liquids in the cartridge, and AFA will remove the wax, extract the DNA, and mix the liquids and reagents in the cartridge. The DNA that AFA removes from samples will also be purified by binding to little magnetic beads, which are held in place by magnets on the machine. This makes the extracted DNA ready for use in other applications, like DNA sequencing. This instrument and cartridge will provide a reliable, reproducible, and labor-saving way to use FFPE samples that are very valuable and sometimes irreplaceable and that can be used for research or by doctors in diagnosing and treating patients. We will do this by breaking down the whole process into small pieces and developing those individually. The pieces are the extraction of DNA using AFA;chemical treatment to remove proteins and to break any chemical bonds between the DNA and itself or remaining protein contamination;and purifying the DNA. The performance of each step will be compared to the manual method. The ways we will measure the performance include determining just how much DNA there is by adding a fluorescent dye and measuring the fluorescence;measuring the length of the DNA molecules (which tells us if they are too broken up to be used for other applications);measuring the contamination by residual protein by seeing how much UV light is absorbed by the DNA solutions;and finally by using a DNA amplification method called quantitative PCR that only works well if the DNA is intact and not bound to other contaminants in the sample. We will also sequence a few samples that have been processed by the prototype. When all of the pieces have been developed individually, they will be put together. For the project, the cartridge to do this work will be mae using a computerized milling or cutting machine that can make high quality models of structures with features of size less than that of the diameter of a human hair. When the cartridge is made into a product it will be made from molded plastic. The instrument will be run by a computer and modified from an existing Covaris AFA instrument. If this project is successful, the next phase will be to solve manufacturing problems, including storing reagents on the cartridge, and scaling the cartridge up to many samples.
Extracting nucleic acids from formalin-fixed paraffin-embedded tissues is vital to cancer research, but it is usually labor-intensive and suffers from low yield and poor quality. We will develop a robust, standardized system to extract and purify nucleic acids from these tissues. A cartridge-based microfluidic system will implement high frequency ultrasonic technology (Adaptive Focused Acoustics), which has already been proven to provide high quantity and quality extractions when used in manual protocols.
