©2006 American Type Culture Collection (ATCC).
DNA typing of human cell lines: historical
perspectiveYvonne Reid, PhDCollection/Research ScientistATCC Cell Biology
©2006 American Type Culture Collection (ATCC).
Outline�• Molecular techniques for the
authentication of human cell lines�• Mechanism of gene duplication�• DNA fingerprinting �– multilocus probes�• DNA profiling �– single locus probes�• DNA profiling �– STR analysis
©2006 American Type Culture Collection (ATCC).
Identification of human cell lines: molecular techniques
�• Interspecies Identification�• Cytochrome Oxidase subunit I (COI)�• Cytogenetics (G-banding)
�• Intraspecies Identification�• RFLP (restriction fragment length polymorphism)
�• Multi-locus probes (DNA fingerprinting)�• Single locus probes (VNTR)
�• AMP-FLP (Amplified fragment length polymorphism)�• STR analysis
�• SNP (single nucleotide polymorphism)
©2006 American Type Culture Collection (ATCC).
Tandem repetitive DNA sequencesDNA Location Degree of
RepetitionNumber of Loci
Repeat Unit Length
Satellite DNA(centromere)
103 to 107 1 to 2 2 to several thousand bp
Minisatellite (multi-locus and single locus probes)(telomere)
2 to several hundred Many thousands
9 to100 bp
Microsatellite (STRs)(randomly scattered)
5 to about a hundred 104 to 105 1 to 6 bp
©2006 American Type Culture Collection (ATCC).
Mechanism of gene duplicationRepetitive sequences or hypervariable
regions are �“hotspot�” for recombination
�• Recombination�• Unequal exchange of sister chromatids�• Unequal exchange of non-sister chromatids
�• Retrotransposition�• Transposition of DNA sequence via an RNA
intermediate
©2006 American Type Culture Collection (ATCC).
Unequal recombination of nonallelic sites(sister chromatids)
1 2 3 4 5
1 2 3 4 5
2 3 4 51
1 2 3 4 5
1 2 3 4 5
1 2 3 4 5
2 3 4 41
1 2 3 5
5
Centromere Repeating unit
**
©2006 American Type Culture Collection (ATCC).
Unequal recombination of nonallelic sites(non-sister chromatids)
1 2 3 4 5
1 2 3 4 5
2 3 4 51
1 2 3 4 5
1 2 3 4 5
1 2 4 5
2 3 3 41
1 2 3 4
5
Centromere Repeating unit
5
*
*
©2006 American Type Culture Collection (ATCC).
Retrotransposition(Transposition of a DNA sequence via RNA intermediate)
Chromosome A Chromosome B
Chromosome A Chromosome B
A 3�’
Transposable Element DNA
mRNA
cDNA
A A A5�’
A A A A 3�’
5�’ 3�’5�’ 3�’
Transcriptionand Processing
T T T T 3�’
Reverse Transcriptase
Reintegration
©2006 American Type Culture Collection (ATCC).
Multilocus probe: 33.6
Jeffreys, A. et al. Nature 314:87-73, 1985
GCTCCAGATTTCGACCTCCACCCGTCCTTCCTG
Myoglobin gene
D D A A
GACCGAGGTCTAAAGCTGGAGGTGGGCAGGAAG
Hinf1 Hinf1
1 32
169 bp Hinf1 fragment
©2006 American Type Culture Collection (ATCC).
Fig. courtesy of © 2000 by Griffiths et al.
Southern Blot analysisDetection p32-labeled single locus or multilocus probe
Isolated DNARestriction digestion
©2006 American Type Culture Collection (ATCC).
DNA fingerprint of 18 cell lines
Probe: 33.6 (p32-labeled)
Restriction enzyme: HaeIII
Gilbert,D., Reid, YA., et al. Am J Hum Genet 45:499, 1990.
8.0 kb
4.0 kb
3.0 kb
2.0 kb
1.0 kb
6.0 kb
©2006 American Type Culture Collection (ATCC).
Properties of single locus probes (VNTR) for DNA profiling
Probe Locus Size Number of alleles
% heterozygosity
pYNH24 D2S44 2.0 kb 40 >97
pCMM86 D17S74 2.2 kb 20 >93pCMM101 D14S13 2.2kb 30 >95
Nakamura, Y., et al. Science 235:1616-1622 (1987)Bever, R., et al. The Second International Symposium on Human Identification, p103 (1991)Nakamura, Y., et al. Nucleic acid Res. 15:10073 (1987)Nakamura, Y., et al. Nucleic acid Res. 16:5222 (1988)Nakamura, Y., et al. Nucleic acid Res. 16:381(1988)Nakamura, Y., et al. Nucleic acid Res. 16:5223(1988)
©2006 American Type Culture Collection (ATCC).
DNA profiling of 6 unrelated cell lines
Single locus probe: D14S13
Restriction EnzymeHinf1
CC
L-18
�™
HTB
-22�™
CC
L-22
1�™
mar
ker
cont
rol
HTB
-19�™
CC
L-11
9�™
CR
L-17
40�™
3.2 kb
1.6 kb
5.7 kb
©2006 American Type Culture Collection (ATCC).
Properties of the STRs for DNA profilingLocus Name
Chromosomal Location
Repeat motif
No of repeating units
Number of alleles observed
D16S539 16q24-gtr GATA 5-15 10
D7S820 7q11.21-22 GATA 6-15 22
D13S317 13q22-q31 TATC 5-15 14
D5S818 5p21-q31 AGAT 7-16 10
CSF1PO 5q33.3-34 TAGA 6-16 15
TPOX 2p23-pter GAAT 6-13 20
VWA 12p12-pter [TCTA] [TCTG]
10-24 28
THO1 11p15.5 TCAT 3-14 20
Amelogenin Not STR, gender determination
Power of discrimination: 1:1.2 X 108
Butler, JM. Forensic DNA typing, 2001
STR analysis
©2006 American Type Culture Collection (ATCC).
EXTRACT DNA
SPOT FTA®PAPER
PCR AMPLIFY SAMPLE
RESOLVE PCR FRAGMENTS
SIZE PCR FRAGMENTS
CONVERT PCR FRAGMENT SIZES TO ALLELES
DATABASE�• Curate�• Global Comparisons�• Lot to Lot Comparisons)
©2006 American Type Culture Collection (ATCC).
Use of allelic laddersConversion of fragment size into alleles
©2006 American Type Culture Collection (ATCC).
STR Polymorphism (homozygous)
M
P
8 repeating units
8 repeating units
AATG
M P
©2006 American Type Culture Collection (ATCC).
STR Polymorphism (heterozygous)
M
P
10 repeating units
9 repeating units
AATGM P
©2006 American Type Culture Collection (ATCC).
STR analysis for human identification
K562
WS1
2 unrelated cell lines
D5S818 D13S317 D7S820 D16S539 vWA THO1 Amel. TPOX CSF1PO
K562 (CML) 11, 12 8 9, 11 11, 12 16 9.3X
female8, 9 9, 10
WS1 (Skin fibro) 13 12 9, 10 10, 11 17, 18 8, 10X
female8, 9 10, 13
©2006 American Type Culture Collection (ATCC).
STR analysis for human identificationTwo related human cell lines
HAAE-2
HFAE-2
D5S818 D13S317 D7S820 D16S539 vWA THO1 Amel. TPOX CSF1PO
HAAE-2aortic artery endothelium
12,13 11,12 8,10 12,13 14,18 7,9X,Ymale
10,11 10,11
HFAE-2 femoral artery endothelium
12,13 11,12 8,10 12,13 14,18 7,9X,Ymale
10,11 10,11
©2006 American Type Culture Collection (ATCC).
BGO1VBG01
STR analysis: parental vs. clone
WiCell ATCC
D5S818 D13S317 D7S820 D16S539 vWA TH01 TPOX CSF1PO Amelogenin
BG01 10, 12 11, 12 10, 11 9, 11 16, 17 7, 9.3 8 10 X, Y
BG01V 10, 12 11, 12 10, 11 9, 11 16, 17 7, 9.3 8 10 X, Y
©2006 American Type Culture Collection (ATCC).
Case of Gender Misidentification
Y pa
intHuman cell line purported to be of female origin
STR
ana
lysi
s G-banding
©2006 American Type Culture Collection (ATCC).
Cellular cross-contamination
©2006 American Type Culture Collection (ATCC).
STR detects 10% contaminants
(A)
(B)
(C)
(A) Uncontaminated CCRF-SB (100%)
(B) CCRF-SB/CCRF-CEM (90%/10%)
(C) Uncontaminated CCRF-CEM (100%)
Characteristics of STR markers
�• High discriminating power�• High observed heterozygosity >70%�• Robust and reproducible results�• Low stutter characteristics�• Low mutation rate�• Alleles fall in the range of 90-500 bps �– smaller
fragments better to allow for degraded DNA.
©2006 American Type Culture Collection (ATCC).
Butler, JM. Forensic DNA typing, 2001
©2006 American Type Culture Collection (ATCC).
Frequency of genetic changes in cell lines
D18S1179 D21S11 D8S1179 FGA THO1 vWF
Heterozygosity of 221 cell lines
59.9% 69.8% 70.3% 69.9% 48.2 61.5
Heterozygosity in Caucasian adults
85.7% 82.7% 82.7% 87.8% 79.1 82.7
Occurrence of 3 alleles 2/177 6/192 4/192 3/183 0/193 5/192
Occurrence of 4 alleles 0 0 0 0 0 1/192
Masters, JR et al. PNAS 98(14):8012-8017, 2001
SGM markers
©2006 American Type Culture Collection (ATCC).
Short tandem repeat (STR)�• Target sequence consists of microsatellite DNA�• Typically use 1 - 2.0 ng DNA (partial degraded) �• 1 to 2 fragments�• Highly informative; 1:10-8 (DNA profiling)�• Banding pattern reproducible�• PCR amplifiable; high-throughput; database�• �“No binning�” to determine fragment size �– allelelic ladders�• Cross-contaminating human cell lines are very easy to detect
and interpret�• Probes are human-specific�• High stringency conditions