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

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

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

Streptavidin – biotin pair polydisperse core-shell polystrene particle 1um mean diameter 1mg SA paricle bind 350pmol free biotin 150pmol biotinylated oligo. ( Kd = 10-15 )

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 >

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

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

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

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

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

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

Result of First training exp. positive < CS,FACULTY,4 > M 1 2 3 4 5 6 7 8 9 10 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

Result of Temp gradient PCR M 1 2 3 4 5 6 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

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

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

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

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

Result of Unknown exp. majority voting <CS,STAFF,4> Before majority voting 1.108 ≒ 39ug/ml Positive selection Negative selection 0.762 ≒ 22.5ug/ml 1.134 ≒ 40ug/ml

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

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