Contamination and misidentification of human tissue culture lines is a significant issue in biological research. It is important that investigators verify the identity of their cell lines periodically to ensure the integrity of their studies. Verification is now often a requirement for both grant funding applications and publication.
The CFG DNA sequencing facility performs human cell line authentication by short tandem repeat (STR) profiling. We use the Promega GenePrint 10 System to generate STR fragments, and fragment analysis is performed with the ABI Prism 3730XL instrument and ABI GeneMapper 4.0 software.
GenePrint 10 System
The GenePrint 10 System from Promega is a PCR-based system for the generation of a multi-locus STR profile from human DNA. The kit provides co-amplification and four-color detection of ten loci, including all eight of the ATCC Standards Development Organization Workgroup's ASN-0002-2011 recommended loci (TH01, TPOX, vWA, CSF1PO, D16S539, D7S820, D13S317, D5S818), D21S11, and Amelogenin for gender determination.
Locus-Specific Information
|
Chromosomal Location | Repeat Sequence | |
| TH01 | 11p15.5 | AATG | |
| D21S11 | 21q11 - q21 | TCTA Complex | |
| D5S818 | 5q23.3 - q32 | AGAT | |
| D13S317 | 13q22 - q31 | TATC | |
| D7S820 | 7q11.21 - q22 | GATA | |
| D16S539 | 16q24 - qter | GATA | |
| CSF1PO | 5q33.3 - q34 | AGAT | |
| Amelogenin | Xp22.1 - 22.3 and Y | NA* | |
| vWA | 12p13.31 | TCTA Complex | |
| TPOX | 2p23 - 2pter | AATG |
* Amelogenin does not contain an STR, but produces a 106-base X-specific band and a 112-base Y-specific band
Human Cell Line Authentication Service
Customers should provide the CFG with high quality genomic DNA obtained from the cell line(s) of interest. Amplifications with GenePrint 10 reagents are then performed at the CFG, and the resulting fragments are separated on our ABI 3730XL and STR profiles generated with GeneMapper software. A report is provided consisting of a gene table with allele calls, fragment sizes, and peak heights for each loci. The report will allow the customer to search publicly available databases to compare results with known genetic profiles.
The following websites host STR databases that can be used for searches:
Pricing
| Service | Academic |
|---|---|
| Human Cell Line Authentication | $100.00 |
Please, contact us about pricing for Non-Academic institutions
To Request Human Cell Line Authentication
1. Customer Information Form
For first time users, complete and submit a Customer Information Form that you can download directly in Adobe Reader format.
This form only needs to be submitted once.
2. Request for Human Cell Line Authetication Form
Complete and submit a Request for Human Cell Line Authetication Form. Please submit additional forms if you are submitting more than 10 samples.
3. Arrange to send samples
Arrange to send genomic DNA sample(s) to the CFG. Please see Template Preparation below for guidelines and appropriate amounts of template to submit.
Guidelines for Submission of Samples for Cell Line Authentication
A. Genomic DNA Template Preparation
The CFG recommends isolation of genomic DNA from cell lines by use of an appropriate commercially available spin-column type kit (i.e., Qiagen, Promega, etc.). Templates submitted should be accurately quantified by determining both A260 and the A260/A280 ratio (ratio must be at least 1.8). Purified genomic DNA should be resuspended or eluted in water or 10 mM Tris/HCl pH 7.5-8.0, not TE, as EDTA will inhibit the activity of the polymerase used for amplification.
The concentration of each genomic DNA sample should be adjusted to 10 ng/ul for submission. We require a minimum of 5 ul per sample. Please submit your samples in 1.5 ml microcentrifuge tubes.
B. Quality Control
All sample amplification reactions are split and run in duplicate wells on the instrument. This helps control for the occasional transient capillary problem, since each well has its own capillary. Before sending out results we compare allele calls for the duplicates, and drop any that have missing allele calls. If at least one has a full set of allele calls, we will provide that duplicate. If the duplicates are identical, we will send just one of the duplicates. Failure of both duplicates, when the control reactions worked as expected, is an indicator of a quality problem with the original genomic DNA sample.
The GenePrint 10 kit provides a control DNA (line 2800M) that we include in every run. We check to make certain that this control produces the expected alleles. We run, in addition, an allelic ladder and a no-template negative control. A report for the 2800M sample is included in the results table that we provide. If the results for your own samples are not what was expected, we can further review the original electropherograms on request.
C. Policy on Repeat Reactions
Because of the quality control measures described above, we do not repeat reactions unless those measures provide clear evidence of an instrument or technical malfunction.