This method yields large quantities of DNA that can easily be collected.

Here’s what you’ll need for each DNA extraction:

Materials Needed:

Raw wheat germ - 1 gram or 1 teaspoon.
Wheat germ can be purchased at a health food store or some large supermarkets; toasted wheat germ does not work.

Liquid detergent - 1 ml or a scant 1/4 teaspoon
The following liquid soap products have been tested and found to work well for this DNA extraction protocol: Lemon Fresh Joy, Woolite, Ivory, Shaper, Arm & Hammer, Herbal Essence shower gel by Clairol, Tide, Dish Drops, Kool Wash, Cheer, Sunlight Dish Soap, Dawn, Delicate, All, and Ultra Dawn.

The following liquid products do not work well: Life Tree, Shout, Shaklee, Sunlight Dishwasher, and LOC. Powdered detergents also do not produce good results with this protocol.

Alcohol - 14 ml or 1 tablespoon
70% isopropyl alcohol (”Rubbing alcohol”) is the least expensive since it can be purchased at a grocery store or pharmacy. However, it contains a higher percentage of water, making it slightly more difficult to precipitate the DNA.

95% ethyl alcohol and Everclear grain alcohol (which is 95% alcohol) both work equally well. The DNA is easy to collect.

50-60° Celsius tap water - 20 ml or 1 tablespoon
Do not use water hotter than 50-60° C. The water will cool during the extraction procedure, but this does not matter. Test your tap water — it may be hot enough right from the tap.

50 ml test tube
Capped test tube, beaker or spice jar.

Graduated cylinder,
Measuring spoons, or other measuring devices.

Wooden applicator stick,
Glass stirring rod/hook, paper clip hook, or shish kebob skewer for stirring and collecting the mixture.

Eyedropper,
Pasteur pipette and bulb, or pieces of paper towel - may be needed to remove foam

Sealable container (optional),
Such as a tube, vial or jar to store DNA.

50% alcohol (optional) - for storing DNA.
You can use either isopropyl alcohol (rubbing alcohol) or ethyl alcohol for storing the DNA you extract. To make 100 ml of 50% alcohol with isopropyl alcohol, mix 71 ml of 70% isopropyl alcohol (rubbing alcohol) with 29 ml of distilled water. Using ethyl alcohol or Everclear grain alcohol, mix 53 ml of 95% ethyl alcohol (ethanol) with 47 ml of distilled water.

Paper towels
or filter paper - for drying DNA

Instructions

1. Place 1 gram or 1 teaspoon of raw wheat germ in a 50 ml test tube, beaker or jar.

2. Add 20 ml or 1 tablespoon of hot (50-60 °C) tap water and mix constantly for 3 minutes.

3. Add 1 ml or a scant 1/4 teaspoon of detergent and mix gently every minute for 5 minutes. Try not to create foam.

4. Use an eyedropper, pipette, or piece of paper towel to remove any foam from the top of the solution.

5. Tilt the test tube, beaker or jar at an angle. SLOWLY pour 14 ml or 1 tablespoon of alcohol down the side so that it forms a layer on top of the water/wheat germ/detergent solution. Do not mix the two layers together. DNA precipitates at the water-alcohol interface (the boundary between the water and the alcohol). Therefore, it is crucial to pour the alcohol very slowly so that it forms a layer on top of the water solution. If the alcohol mixes with the water, it will become too dilute and the DNA will not precipitate.

6. Let the test tube, beaker or jar sit for a few minutes. White, stringy, filmy DNA will begin to appear where the water and alcohol meet. You will usually see DNA precipitating from the solution at the water-alcohol interface as soon as you pour in the alcohol. If you let the preparation sit for 15 minutes or so, the DNA will float to the top of the alcohol.

   You can usually get more DNA to precipitate from the solution by using one of the DNA-collecting tools (such as a glass or paper clip hook) to gently lift the water solution up into the alcohol. This allows more DNA to come in contact with the alcohol and precipitate. You may find it helpful to pour the water/detergent solution into a clean test tube, leaving behind the wheat germ, before adding the alcohol.

7. Use a glass or paper clip hook or a wooden stick to collect the DNA.

8. If you want to keep the DNA, store it in 50 - 70% alcohol in a sealed tube or air dry it on paper towels or filter paper.

Maximize Quality and Quantity of DNA from Mailed Cytobrushes
As part of the mission to prevent birth defects, NCBDDD has established the National Birth Defects Prevention Study (NBDPS), a multi-site, population-based case-control study to identify potential genetic and environmental risk factors for birth defects. The study includes ascertainment of case and control infants, maternal interviews, and collection of DNA from cheek cells using cytobrushes mailed to each mother, father, and infant. To identify potential genetic risk factors, CDC and collaborating sites have collected buccal-derived DNA samples for 9,140 case-infants, 10,465 case-infant mothers, 8,703 case-infant fathers, 3,346 control-infants, 3,377 control-infant mothers, and 2,802 control-infant fathers. DNA is extracted from self-collected cytobrushes and stored for later analysis. One problem encountered in the NBDPS is that the extracted DNA is sometimes of insufficient quality and quantity for use with current whole genome amplification (WGA) methods. WGA is necessary for high-throughput genotyping of buccal-derived DNA samples. Other projects that include self-collection of buccal samples using cytobrushes may be experiencing the same problem with less than optimal quality and quantity DNA.
Phase I will use mailed cytobrush samples that are not part of the NBDPS.
1. Develop a WGA method that provides optimal DNA quantities from mailed cytobrush samples, both new and existing.
2. Identify a feasible, innovative technology that allows laboratories to extract DNA of optimal quantity and quality on new samples collected from buccal brushes.
The new technology will increase the number of DNA samples that are of useable quality and quantity for further analyses.
The new technology will allow maximum utilization of existing biologic samples, minimize the intrusion on study participants, and save funds in biologic sample recollection cost.

Antibody Array for Cancer Detection

Number of anticipated awards: 3-5
(Fast-Track proposals will not be accepted.)
Budget (total costs): Phase I: $150,000;
Phase II: $1,000,000

The deadline for receipt of all contract proposals submitted in response to this solicitation is: 5:00 p.m. Eastern Standard Time Monday, November 5, 2007

The purpose of this initiative is to develop high throughput antibody arrays for quantitative analysis of multiple biomarkers for early detection and diagnosis of cancer. These arrays may include antibodies based on the applicant’s own research and knowledge of the literature. Applicants are also encouraged to contact extramural investigators from the NCI’s Early Detection Research Network (EDRN; www.cancer.gov/edrn), which is developing a number of biomarkers for cancer detection, diagnosis and prognosis. Please contact one of the members or associate members at: http://edrn.nci.nih.gov/memberslist/x.xml. The selected applicants will develop microarrays, the chemistry of which may be based on nanotechnology and/or microfluidics. Applicants should focus initial development on the diagnosis and early detection of prostate, breast, lung, colon, and other major epithelial cancers. In Phase II, the antibody microarray developed in Phase I will be validated under a plan developed with the NCI project officer. Applicants are encouraged (but not required) to develop a validation plan that includes the participation of EDRN investigators. It is anticipated that such participation will result in accelerated development, production, validation and commercialization of antibody microarray technology for early detection and diagnosis of cancer.

Project goals:

* Prepare and purify biomarker-specific antibodies in the form of recombinant antibodies or monoclonal antibodies (mAb) and construct arrays.
* Develop and/or improve methodologies for quantitative measurements of the bound antigens on Ab microarrays.
* Perform analytical validation, e.g., test the reproducibility, sensitivity, specificity and dynamic range of detection in collaboration with EDRN and other institutions to measure the efficacy of the developed array.

Currently, there is no single marker or a combination of biomarkers that has sufficient sensitivity and specificity to diagnose early stage cancer. However, recent developments in gene and proteomic profiling of precancerous and cancerous lesions suggest that a combination or a patterns of markers may be used to distinguish between cancer and non-cancer with high sensitivity and specificity (95-100%). Innovative technologies, such as microfluidics and nanotechnology, combined with antibody arrays are likely to provide a reliable, sensitive and quantitative detection tool for measuring differentially expressed biomarkers from a limited amount of sample (20ul or less of serum). The involvement of small businesses through the SBIR contract mechanism will strengthen the EDRN’s efforts in the development, validation and commercialization of biomarkers for early detection and risk assessment.

The work performed under this contract is between a selected Biotech Company and EDRN Investigator/s and the NCI EDRN Program staff. Unless otherwise agreed to in writing between the selected Biotech Company and the institutions of the EDRN, the following will guide the Intellectual Property management and sharing of research resources generated from the work performed under this contract. All inventions conceived or first actually reduced to practice solely by the selected Biotech Company investigators under this Agreement will be the property of the selected Biotech Company in accordance with 35 USC Section 200, et. seq., subject to any intellectual property (IP) rights of the providers of biomarkers (e.g., institutions of EDRN investigators) to the selected Biotech Company. All inventions conceived or first actually reduced to practice jointly by the selected Biotech Company and any EDRN Investigators or NCI EDRN program staff will be jointly owned by the inventors’ institutions. The Selected Biotech Company agrees that it will permit EDRN Investigators to use such inventions under terms consistent with the Principles for Recipients of NIH Research Grants and Contracts on Obtaining and Disseminating Biomedical research Resources (http://grants.nih.gov/grants/intell-property_64FR72090.pdf).

The providers of biomarkers will retain their respective IP rights for those biomarkers developed by their institution’s respective investigators. The selected Biotech Company is responsible for negotiating access rights, including any commercial license rights, to all materials provided to the selected Biotech Company, and any related IP, in order to conduct the activities funded under this Agreement.

NCI, EDRN Investigators and the EDRN Data Management and Coordinating Center, managed by the Fred Hutchinson Cancer Research Center (“EDRN DMCC”) will have unlimited rights as defined in FAR 52.227-14, general to the following data developed during the course of this project: (i) protocols for using the antibody microarrays to be developed by the selected Biotech Company and individual antibodies independent of the antibody arrays; (ii) initial research results concerning the use of antibodies against specimens provided by EDRN Investigators; (iii) antibody characterization data, including the results of testing to demonstrate the utility of particular antibodies; and (iv) validation data based on subsequent experiments involving the antibody microarrays.

Authorship of publications resulting from data developed under this contract will be shared by all contributing parties including the NCI.

Phase I Activities and expected deliverables:

Relevant biomarkers could be selected from published literature or by contacting one of the EDRN extramural investigators. (Please contact one of the members or associate members at: (http://edrn.nci.nih.gov/memberslist/x.xml.)

* Establish the proof of principle.
* Develop an antibody microarray for detection of 3 biomarkers using innovative technologies.
* Demonstrate that the tiled antibodies perform as well or better than a conventional ELISA in the detection of these biomarkers in serum from cancer patients.

Phase II Activities and expected deliverables:

Applicants are encouraged (but not required) to develop a validation plan that includes the participation of EDRN investigators.

* Develop antibody microarrays with a capability to simultaneously detect and measure the concentration of 30-50 biomarkers. These arrays can include both biomarkers from the EDRN as well as biomarkers identified by the small business.
* Validate antibody microarrays in high-risk subjects.
* Produce and test up to 1000 microarrays with samples from normal and case subjects.