28/10/98 Mayer et al., Serono Pharmaceutical Research Institute p.1/8. A Very Large Scale, High Throughput and Low Cost. DNA Sequencing Method based on ...
A Very Large Scale, High Throughput and Low Cost DNA Sequencing Method based on a New 2-Dimensional DNA Auto-Patterning Process
P. Mayer, (L. Farinelli), G. Matton, C. Adessi, G. Turcatti, J.J. Mermod, E. Kawashima. Genomic Technology Department Serono Pharmaceutical Research Institute
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3’ end free in solution
5~10nm
Primers p1 + p2
Covalent attachment
0.3 ~ 2 cm
Prepared sample 1 Prepared sample 2 . . . Prepared sample n 28/10/98
~1kb => ~300nm > persistence length
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Anneal
Elongate
Separate strands wash
< d > = f ( [PS] ) INITIATE
Cycle 1 h < 300 nm
Cycle 2
...
< s > = f ( number cycles )
Cycle n
=> DNA colonies 28/10/98
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Experimental conditions Prepared samples :
T1 T2
Support : NucleolinkTM tubes covered with red and green oligos 3mm
Colony formation with different mixes of T1 and T2 Probe with biotinylated nick-translation DNA probes and
40x
fluorescent streptavidin-beads (40nm)
Setup : inverted epi-fluorescence microscope with 40x objective, cooled CCD camera, computer
CCD 28/10/98
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2~10 µm
DNA colonies
1~2µm
samples : 100% T1, 0% T2 10µm
samples : 0% T1,100% T2
10µm
Hybridization with probe specific to T1
=> Number of spots ∝ samples
=> 1~10 million samples / cm2 samples 10% T1,90% T2 28/10/98
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10µm
Sequencing : stepwise primer extension using DNA polymerase and fluorescent nucleotides
Fluorescence increase on individual DNA colonies A AA
Primer Template
A
A
A Wash + Image analysis
AC
AAC
Wash + Image analysis
C
C
T
n cycles
AACG
Wash + Image analysis AG Wash + Image analysis
G
in average, 2 bases read / cycle 28/10/98
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CG
222 304
264 394
215
212
+BT
+ AC
..TGACT seq 1 : TATACTGACCT
+ CC
Cy5 labeled
..TGAT seq 2 : GCTACTATTCT 267 383
217
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Objectives : ~10 bases = tag counting, genotyping >20 bases = “sequencing” Mayer et al., Serono Pharmaceutical Research Institute
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Extrapolating from current experimental results : EXPECTED THROUGHPUT Ultimate limiting factors, presently : time to acquire an image, ~10s colony density, 10,000 colonies/image (20x objective, 2kx2k CCD)
raw average => 5000 bases/10s -> usable :
50~500 bases/s
(“ABI” 1~4 base/s)
EXPECTED COSTS : ~$1000 for 2 day operation on 18x18 mm “chips”
-> 6.106-7 bases raw sequence => $1 = 6,000~60,000 bases (“ABI” 3~10 base/$) (optimistic : 10x objective, 5.104 colonies/image -> x20 longer term : 1s exposure -> x200) 28/10/98
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17 bases
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Pharmacogenomics: relating genotype to drug response
Many polymorphism... in many genes... in many patients !!!
=> Giga
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base projects Mayer et al., Serono Pharmaceutical Research Institute
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Assay monitoring
Sample collection
Sequence analysis Sample assaying
Sample preparation & arraying 28/10/98
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Assay monitoring
Genomic Technology Dpt. :
Sample assaying
“Nano/micro technology” based on biochemical auto-patterning avoiding (micro-)robotics & micro-lithography
Sample preparation & arraying 28/10/98
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