1. Version Space learning with DNA Molecules
Hae Man . Jang
proteome Lab
Hanyang Univ

2. History of experiment Sequence design using NACST
Oligo synthesis Bioneer
Generation of all hypothesis confirmed by gel
Bead separation
using CPG , MPG LCA covalent bond
failed to separation , unexpected result
Introduction fluorescence tagged oligo seq. for majority voting
generated quantitative table using intensity
Change bead system
Roche , streptavidin Magnetic particle

3. MPG LCA covalent bond 15 A aliphatic arm give flexibility
loading 75~125nmole / mg MPG

4. Streptavidin – biotin pair
polydisperse core-shell polystrene particle
1um mean diameter
1mg SA paricle bind
350pmol free biotin
150pmol biotinylated oligo.
( Kd = )

5. Procedure Generation of all hypothesis
Make Quantitative table for majority voting
Produce magnetic probe for +/- selection
First training exp positive
< cs,faculty,four >
Second training exp negative
< cs,staff,five >
Unknown exp majority voting
< cs,staff,four >

6. Generation of all hypothesis
95oC 5min denaturation
Temp decrease 1oc/1 cycle
ligation
3% native gel cs
cs’
faculty
faculty’
four
four’ ee
ee’
staff
staff’
five
five’
?dept
?dept’
?status
?status’
?floor
?floor’
100bp
75bp
50bp
25bp

7. Make Quantitative table for majority voting ; at 260nm UV spec.

8. Make Quantitative table for majority voting ; at 516nm emission spec.

9. Produce magnetic probe for +/- selection
1mg SA magnetic particle uptake
Bead activation
wash 3 times with 10mM Tris-HCl,1mM EDTA, 100mM NaCl
Probe attach to Bead
incubation 10min / 30min / 2h / overnight
with 10mM Tris-HCl,1mM EDTA, 100mM NaCl
Washing
wash 2 times with 10mM Tris-HCl,1mM EDTA, 1M NaCl
Confirm
using UV spec. at 260nm analyze DNA con. in supernatant

10. First training exp. positive < CS,FACULTY,4 >
Uptake 200ul of initial pool
Add 50ul each of <CS>, <Don’t Dept> magnetic probe
Denature 95 C 5min
Hybridization 2h at room temp.
Washing 2 times with D.W
Uptake supernatant
Sequentially add < FACULTY / Don’t STAT > , < 4 / Don’t floor > and process
Conform by PCR

11. After First training exp. positive < CS,FACULTY,4 >
<cs, faculty, four> <cs, faculty, ?> <cs, ?, four> <?, faculty, four> <cs, ?, ?> <?, faculty, ?> <?, ?, four> <?, ?, ?>
Total 8 hypothesis

12. Result of First training exp. positive < CS,FACULTY,4 >
M M
100bp
75bp
50bp
25bp
M : 25bp marker (KDR)
lane 1: cs / 4
lane 2: cs / don’t floor
lane 3: don’t dept / 4
lane 4: don’t dept / don’t floor
lane 5: ee / 5
lane 6: negative control
lane 7: ligation mixture
lane 8: sup. after cs/don’t dept
lane 9: sup. after faculty /don’t stat
lane 10: sup. after 4/don’t floor
Figure 1. PCR product of 1st positive selection
was confirmed 3% Agarose gel electrophoresis

13. Result of Temp gradient PCR
M M
100bp
75bp
50bp
25bp
50C
54C
56C
M : 25bp marker (KDR)
lane 1: cs / 4
lane 2: cs / don’t floor
lane 3: don’t dept / 4
lane 4: don’t dept / don’t floor
lane 5: ee / 5
lane 6: negative control
Figure 2. Temp gradient PCR product of 1st
positive selection was confirmed 3% Agarose
gel electrophoresis

14. Second training exp. negative <CS,STAFF,5>
Add 30ul each of <EE>, <Facultyt>, <4> Magnetic probe at same time
Hybridization 2h at room temp. with gentle agitation
Washing 2 times with D.W
Denature 95 C 5min
Uptake supernatant

15. After Second training exp. negative < CS,STAFF,5 >
<cs, faculty, four> <cs, faculty, ?> <cs, ?, four> <?, faculty, four> <?, faculty, ?> <?, ?, four>
Total 6 hypothesis

16. Unknown exp. majority voting < CS,STAFF,4 >
Expected seq. after positive / negative selection
<cs, faculty, four> <cs, faculty, ?> <cs, ?, four> <?, faculty, four> <?, faculty, ?> <?, ?, four>
if <cs, staff,4> is unknown ,
positive (n) = <cs,?,four> , <?,?,four>
negative (u) = <cs, faculty, four>,<cs, faculty, ?>
<?, faculty, four>, <?, faculty, ?>
(-) : (+) = 4 : 2

17. Unknown exp. majority voting <CS,STAFF,4>
Measure Ab. at 260nm of sample which followed positive / negative selection ( 200ul )
Divide sample to each 100ul vol.
positive selection
<cs , don’t dept>
<staff, don’t stat>
< 4 , don’t floor>
negative selection
<ee>/ <faculty>/ <5>
3. Measure Ab. at 260nm of each sample
4. PCR
5. Electrophoresis

18. Result of Unknown exp. majority voting <CS,STAFF,4>
Before majority voting
1.108 ≒ 39ug/ml
Positive selection
Negative selection
≒ 22.5ug/ml
1.134 ≒ 40ug/ml

19. Result of Unknown exp. majority voting <CS,STAFF,4>
100bp
M p1 p2 p3 p4 n1 n2 n3 n4 n5
75bp
50bp
25bp
M : 25bp marker (KDR)
lane 1: cs / 4
lane 2: don’t dept / 4
lane 3: ee / 5
lane 4: negative control
lane 5: cs / 4
lane 6: cs / don’t floor
lane 7: don’t dept / 4
lane 8: don’t dept / don’t floor
lane 9: negative control
Figure 3. PCR product of majority voting
was confirmed 3% Agarose gel electrophoresis

20. Discussion We proposed an novel method to implement version space
learning with DNA
We used UV / fluorescence to measure DNA in Majority voting
There are some false positive in magnetic separation ,
but it is overcame using hard washing condition such as
temp,NaOH..
For more sensitive separation with magnetic probe
Above 1M NaCl concentration in binding Buffer
Need spacer seq. in biotinylated oligo probe
Biotin---(A)25-nnnnnnnnnnnnnnn