Specimen Preparation for In Situ PCR

Award Information
Agency: Department of Health and Human Services
Branch: N/A
Contract: 1 R43 CA65307-1,
Agency Tracking Number: 24915
Amount: $75,000.00
Phase: Phase I
Program: SBIR
Awards Year: 1994
Solicitation Year: N/A
Solicitation Topic Code: N/A
Solicitation Number: N/A
Small Business Information
Grace Bio-oncology Laboratoryo
900 Auburn Road, Suite 105, Pontiac, MI, 48342
DUNS: N/A
HUBZone Owned: N
Woman Owned: N
Socially and Economically Disadvantaged: N
Principal Investigator
 Charles Mc Grath
 (313) 332-7100
Business Contact
Phone: () -
Research Institution
N/A
Abstract
Our objective is to increase the sensitivity of in situ PCR , by improving methods of specimenpreparation. We have devised a LYOPHILM technology, which comprises lyophilization of specimen onspecialized microporous nitrocellulose films, to allow reactants to thoroughly penetrate specimen tomaximize reactant access to template, under conditions which do not consequently redistribute templateand amplified product within the specimen. In Phase I, we propose to validate LYOFlLM as a single steppreservation method, by head-to-head comparison with a "conventional" preservation method whichemploys three separate chemical steps of fixation (vis. paraformaldehyde), dehydration (vis. alcoholseries) and permeation (vis. proteolysis). We will use b-actin primer pairs selected to provide multi-levelnon-specific controls to determine the most sensitive specimen preparation format for both DNA andmRNA (cDNA) PCR in thin sections of breast cancer. We will then use the most sensitive method todetermine ability to detect 1-3 copies of p53 sequences in nuclear DNA of cloned prostate cancer cells.We will use digoxygenin-conjugated nucleotide incorporation and FlTC-conjugated antibody detectionmethods to quantitatively measure sensitivity of in situ PCR. In Phase 2, we will optimize conditionsto maximize sensitivity and accuracy; and develop the technology into a standard method suitable forquantitative cell analysis in automated format.

* information listed above is at the time of submission.

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