Date of Award

12-1-2014

Degree Type

Thesis

Degree Name

Master of Science

Field of Study

Biomedical Sciences

Department

Graduate School of Biomedical Sciences

First Advisor

Patricia A. Gwirtz

Abstract

Human cell lines are extensively used in scientific research. Cell line contamination can occur anytime during a research project due to exposure to another human cell line. Thus, authentication of human cell lines is recommended multiple times during research. The Short Tandem Repeat (STR) analysis method has been used to authenticate human cell lines. STR is a complex process involving several steps, such as DNA extraction and purification. To make this identification method simple and quick, manufacturers have been manufacturing kits that can amplify the DNA directly from cells spotted on a storage medium without DNA extraction and purification. Many laboratories offer the direct cell line authentication service at a high cost due to these laboratories using expensive kits and storage media. In this practicum study, the Human ID Bloodstain Cards (GE Healthcare Life Sciences, Piscataway, NJ) and the GenePrint® 10 System Kit (Promega Corp., Madison, WI) were used to establish a cost-effective direct STR amplification method. Three concentrations of DU-145 cell suspension solutions (5000 cells/μL, 12,500 cells/μL, and 25,000 cells/μL) were spotted onto a Human ID Bloodstain Card, and two card punches of 1.2 mm diameter were taken from each concentration for analysis. The American Tissue Culture Collection (ATCC, Manassas, VA) established the ASN-0002 standard for cell line authentication, which recommends amplification of a minimum of eight autosomal loci and sex determining loci (Ameleogenin). The results showed that the STR profiles obtained from the Human ID Bloodstain Card samples using the GenePrint® 10 System kit met the ASN- 0002 standards.

Comments

Vikram, Vemireddy. Authentication of Human Cell Lines and Detection of Human Cell Line Contamination by Cost-Effective Direct Amplification of DNA. Master of Science (Biomedical Sciences, Biotechnology). November, 2014.

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