dna typing of human historical perspective · dna typing of human historical perspective phd...

©2006 American Type Culture Collection (ATCC).

DNA typing of human cell lines: historical

perspectiveYvonne Reid, PhDCollection/Research ScientistATCC Cell Biology


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


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)


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


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


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

**


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

*

*


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


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


Fig. courtesy of © 2000 by Griffiths et al.

Southern Blot analysisDetection p32-labeled single locus or multilocus probe

Isolated DNARestriction digestion


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


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)


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


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


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)


Use of allelic laddersConversion of fragment size into alleles


STR Polymorphism (homozygous)

M

P

8 repeating units

8 repeating units

AATG

M P


STR Polymorphism (heterozygous)

M

P

10 repeating units

9 repeating units

AATGM P


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


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


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


Case of Gender Misidentification

Y pa

intHuman cell line purported to be of female origin

STR

ana

lysi

s G-banding


Cellular cross-contamination


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.


Butler, JM. Forensic DNA typing, 2001


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


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

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