Greg Sykes, Robyn Hedges,
Ming Hui, and Yvonne Reid
STR Analysis: From Cells To Data
Cultured Cells
Import Into In-House
Database for Comparison
(Paradox macro)
DNA Extraction
(MoBio UltraClean DNA
Bloodspin Kit, FTA Paper)
GeneScan 3.1 and
Genotyper 2.0 Analysis
(peak detection and labeling)
PowerPlex 1.2 PCR
(Promega)
Electrophoretic
Fractionation and
Fragment Detection
(ABI 310)
Table 1. The Promega PowerPlex 1.2 STR Loci
STR Locus
 Amelogenin
 CSF1PO
 D5S818
 D7S820
 D13S317
 D16S539
 TH01
 TPOX
 vWA
Chromosomal Location
Xp22.10-22.3 and Y
5q33.3-34
5q21-q31
7q
13q22-q31
16q24-qter
11p15.5
2p23-pter
12p12-pter
5’3’ Repeat
n/a
AGAT
AGAT
AGAT
AGAT
AGAT
AATG
AATG
AGAT
Probability of a random match is lower than 1 in 108.
ATCC holds over 2,000 human cell lines.
Cell Line Workflow and How It
Relates To the STR Analysis
Depositor Material/
Token Freeze
Master Cell Bank
(Seed Freeze)
Working Cell Bank
(Distribution Freeze)
Profile Baseline
• Designated as oldest, most
original material
• Cross-compared against all
existing profiles at ATCC
Comparison Profiles
• Cross-compared against all
profiles of that cell line
Unlike STR profiles generated from normal
human material, valid data from tumor cell
lines are more complex due to:
• Heterogeneous original population (differences between
cancer cells within the same mass) and subsequent
selection.
• Addition or loss of chromosomal material due to uneven
karyokinesis and cell hybrids.
• Mutation events occurring within the DNA sequence.
• Genetic drift with subsequent culture expansions.
Figure 1. Example of a standard karyotype from
CCL-75 (WI 38), normal lung tissue (n=46)
Figure 2. Example of a karyotype from CRL-2061
(SJRH30), a rhabdomyosarcoma cell line (n=84)
Figure 3. This relatively balanced-peak electropharogram of a
colon adenocarcinoma cell line is atypical of cell line profiles.
Figure 4. Most tumor and transformed cell line profiles have
unbalanced peaks, but the data are reproducible. This example
happens to be from a liver adenocarcinoma.
Figure 5. This sample was submitted to the ATCC as a cancer
line coming from a single patient. With six loci having trisomies
or more, this line is considered cross contaminated.
The amelogenin locus is useful in cell
culture profiles:
• Finding appropriate X or X, Y peaks confirms the
gender of the cell line (when provided).
• Female-derived cell lines with X, Y profiles are
investigated; if the Y cannot be explained, the line is
failed (not accessioned).
Results From Chromosome Rearrangement
Y Chromosome
Figure 6. Based upon classic cytogenetic testing, cell line
HTB-144 (JAR) was labeled “female.” STR analysis detected
“Y” amelogenin. FISH analysis confirmed translocated Y
chromosome material.
Cell lines submitted as male but lack
Y amelogenin are documented and
approved because:
• A deletion on the Y chromosome amelogenin primer
binding site may prevent a strong (or any) signal.
• Certain cancers (ex. bladder, renal, prostate, stomach,
some leukemias) are more prone to a loss of the Y
chromosome.
• Y chromosome is absent in up to 85% of the bone
marrow cells of normal elderly males.
Parental
Figure 7. Profile differences may emerge between normal
tissue and tumors from the same patient or between a
parental and derived cell lines. These differences may
include:
• Nothing
• Loss of heterozygosity (including locus drop-outs)
• Gain of heterozygosity (somatic mutations)
Parental:
scrape
Derived:
trypsin
Figure 7. Profile differences may emerge between normal
tissue and tumors from the same patient or between a
parental and derived cell lines. These differences may
include:
• Nothing
• Loss of heterozygosity (including locus drop-outs)
• Gain of heterozygosity (somatic mutations)
Parental
Derived
Figure 8. Compared to the parental cell line CRL-2570 (A3), a
microvariant emerges in a derived line, CRL-2571 (I 9.2),
following exposure to the frame-shifting mutagen, ICR-191.
Unstable Alleles
A
B
C
D
P13
P13
P11
P13
E
F
G
H
MASTER
P8
P14
P13
P14
I
J
K
L
P15
P13
P13
P11
Figure 9. Cell lines rarely exhibit allelic instability. This
umbilical cord line, CRL-1730 (HUV-EC-C), is an exception.
Allele 9 at D13S317 has different intensities throughout the
individual lots. However, the intensity of allele 9 within each
lot is reproducible (ex. lot A = weak 9; lot B = strong 9; lot D =
absent 9). Pn is the passage number.
Post-Electropherogram Interpretation
and Data Analysis
• Computer analysis cross-compares new baseline data with
all previously generated profiles.
• Working stock profiles are imported and checked against all
earlier profiles of that cell line.
• Cell lines with at least two independent analyses are posted
on the ATCC website (http://www.atcc.org/Products/str.cfm).
• Commercially available kits enable research institutions,
collections, patent offices, and scientists to confidently
confirm or dispute cell line purity and authenticity.
Summary
• Prior to their accession and distribution, ATCC uses
STR analysis to screen all human cell lines for
authenticity and purity.
• Tumor cell lines are unlike healthy tissue living within
an organism. Most cell lines have undergone genetic
mutation events. Abnormal karyology and sequence
mutations impact the STR profile.
• Tumor and transformed cell line data interpretation is
more challenging than that from normal material, but
the data are reproducible.
• Resulting STR data can be globally communicated to
researchers.
Acknowledgements
Robyn Hedges  Ming Hui  Yvonne Reid
tech@atcc.org
Ed Cedrone
Scott Durkin
Brett Hankins
Qassim Azizi
Kristen Mundy
Ruth Monk
The most exciting phrase to hear in science,
the one that heralds new discoveries, is not
"Eureka!" (I found it!) but "That's funny."
—Isaac Asimov