Rapid and Efficient PCR Cleanup Filters

Award Information
Agency:
Department of Health and Human Services
Branch
n/a
Amount:
$717,229.00
Award Year:
2009
Program:
STTR
Phase:
Phase II
Contract:
2R42RR024968-02
Award Id:
89572
Agency Tracking Number:
RR024968
Solicitation Year:
n/a
Solicitation Topic Code:
n/a
Solicitation Number:
n/a
Small Business Information
DIFFINITY GENOMICS, INC., 150 LUCIUS GORDON DR, WEST HENRIETTA, NY, 14586
Hubzone Owned:
N
Minority Owned:
N
Woman Owned:
N
Duns:
611509451
Principal Investigator:
LEWISROTHBERG
(585) 273-4725
ROTHBERG@CHEM.ROCHESTER.EDU
Business Contact:
LEWISROTHBERG
() -
rothberg@chem.rochester.edu
Research Institute:
UNIVERSITY OF ROCHESTER

UNIVERSITY OF ROCHESTER
518 Hylan Bldg., Box 270140
ROCHESTER, NY, 14627 8047

Nonprofit college or university
Abstract
DESCRIPTION (provided by applicant): Analysis of DNA is important in many applications including forensics, diagnostic genetic testing and biomedical research. Genomic DNA analysis uses chemical amplification methods such as polymerase chain reaction (PCR ) amplification of a target sequence to increase the amount of the genetic fragment under study. This is essential for sequencing which promises to have enormous impact on medicine and will be a cornerstone in personalized medicine where therapeutic cour ses are linked to our genetic makeup. PCR, however, is only an early step for important genomic assays such as capillary sequencing and clean up of the PCR product is required to remove unamplified primers and excess nucleotides (dNTP). Cleanup is a sign ificant contribution to the time, cost and labor involved in DNA analysis. Diffinity Genomics has licensed technology developed in the PI's lab at the University of Rochester that is promising for simple, inexpensive cleanup of PCR product. The process req uires less than 1 minute, an order of magnitude less than present approaches. The PCR solution is exposed to functionalized nanoporous silica with surfaces designed to adsorb dNTP and unamplified primers but not PCR amplicons. The method leaves the desired analyte in solution and removes the impurities so it is faster and cleaner than spin columns where one adsorbs all of the materials onto a support by adding chaotropic salts and selectively elutes PCR amplicons off the support. In our Phase I project, we demonstrated that the efficacy of our process for removing dNTP and retaining amplified DNA is comparable to that of the conventional methods and that using the cleaned up DNA produces equally good sequencing results. We also showed that the functionalized surfaces could be retained in pipette tips so that the cleanup process is reduced simply to aspirating and dispensing the PCR solution. Beta testers were able to clean up their samples ten times faster than with their current cleanup method and achieve go od sequencing results. One goal of the present proposal is to produce 10,000 prototype pipettes for PCR cleanup. We will demonstrate manufacturability and develop additional analytical methods to characterize our materials and process. We will continue tes ting of a variety of PCR conditions and expand our beta testing program to ascertain robustness of the protocol and develop product performance claims. PUBLIC HEALTH RELEVANCE: Individual genetics will increasingly be used both to prescribe therape utic courses and to develop personalized drugs so that screening and sequencing of DNA are very important to medical research and diagnosis. Polymerase chain reaction (PCR) is used to increase the amounts of target sequences in DNA and is routinely used in most DNA analysis but requires lengthy and cumbersome cleanup to remove unwanted materials from solution prior to sequencing. We propose to commercialize filters in a pipette tip that dramatically reduce the time, cost and labor associated with the cleanu p procedure.

* information listed above is at the time of submission.

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