What is an Oligo?What is an Oligo?H d I k ?H d I k ?How do I make one?How do I make one?

(And why should I care?)(And why should I care?)

Oligo: OligonucleotideOligo: OligonucleotideOligo: OligonucleotideOligo: Oligonucleotide

Oligo is the Greek prefix for ‘having few’ orOligo is the Greek prefix for ‘having few’ orOligo is the Greek prefix for having few or Oligo is the Greek prefix for having few or ‘having little’‘having little’Nucleotide is the singular form of NucleicNucleotide is the singular form of NucleicNucleotide is the singular form of Nucleic Nucleotide is the singular form of Nucleic Acid.Acid.

M13F(-20) : 5’-TGT AAA ACG ACG GCC AGT-3’

History of Oligo synthesisHistory of Oligo synthesisHistory of Oligo synthesisHistory of Oligo synthesis

19531953 –– Watson & Crick discover theWatson & Crick discover the1953 1953 Watson & Crick discover the Watson & Crick discover the structure of DNA structure of DNA –– Nobel PrizeNobel Prize19551955 HH phosphonate and phosphatephosphonate and phosphate1955 1955 -- HH--phosphonate and phosphate phosphonate and phosphate triester methods of oligonucleotide triester methods of oligonucleotide synthesissynthesis Slow low efficiencySlow low efficiencysynthesis. synthesis. –– Slow, low efficiency.Slow, low efficiency.

History of Oligo synthesisHistory of Oligo synthesisHistory of Oligo synthesisHistory of Oligo synthesis

19581958 -- Phosphodiester synthesisPhosphodiester synthesis –– SlowSlow1958 1958 Phosphodiester synthesis Phosphodiester synthesis Slow, Slow, length restricted. Requiring enzymatic length restricted. Requiring enzymatic stitching together to make oligosstitching together to make oligosstitching together to make oligos.stitching together to make oligos.

History of Oligo synthesisHistory of Oligo synthesisHistory of Oligo synthesisHistory of Oligo synthesis

19691969--19781978 -- Phosphotriester synthesisPhosphotriester synthesis19691969 1978 1978 Phosphotriester synthesis. Phosphotriester synthesis. Highly efficient. First use of solid support.Highly efficient. First use of solid support.

Interlude: What is solid support?Interlude: What is solid support?Interlude: What is solid support?Interlude: What is solid support?

History of Oligo synthesisHistory of Oligo synthesisHistory of Oligo synthesisHistory of Oligo synthesis

19811981--19841984 -- Phosphite triester synthesisPhosphite triester synthesis19811981 1984 1984 Phosphite triester synthesis. Phosphite triester synthesis. Modern oligo synthesis. Essentially Modern oligo synthesis. Essentially unchanged to dateunchanged to dateunchanged to date.unchanged to date.

History of Oligo synthesisHistory of Oligo synthesisHistory of Oligo synthesisHistory of Oligo synthesis

19831983 –– PCR & a Nobel PrizePCR & a Nobel Prize1983 1983 PCR & a Nobel PrizePCR & a Nobel Prize

Production ProcessProduction ProcessProduction ProcessProduction Process

SynthesisSynthesisSynthesisSynthesisDeprotection/CleavageDeprotection/CleavageQCQCQCQCLiquid HandlingLiquid Handling

Production ProcessProduction Process -- SynthesisSynthesisProduction Process Production Process SynthesisSynthesis

Preparation of Reagents Water is yourPreparation of Reagents Water is yourPreparation of Reagents. Water is your Preparation of Reagents. Water is your enemy.enemy.Cycle SynthesisCycle SynthesisCycle SynthesisCycle Synthesis

Interlude: Effects of Coupling Interlude: Effects of Coupling Effi iEffi iEfficiencyEfficiency

99.5% Coupling Efficiency

90 0%

100.0%

30 0%

40.0%

50.0%

60.0%

70.0%

80.0%

90.0%

nt Full Length Material

0.0%

10.0%

20.0%

30.0%

1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96 101

Length

Percen

98% Coupling Efficiency

70.0%

80.0%

90.0%

100.0%

Material

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%

Percent Full Length M

0.0%

1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96 101

Length

Production Process Production Process ––D i /ClD i /ClDeprotection/CleavageDeprotection/Cleavage

Remove protecting groups & cleave oligoRemove protecting groups & cleave oligoRemove protecting groups & cleave oligo Remove protecting groups & cleave oligo from solid support.from solid support.

Interlude: Protecting groupsInterlude: Protecting groupsInterlude: Protecting groupsInterlude: Protecting groups

Production ProcessProduction Process -- QCQCProduction Process Production Process QCQC

UV260 QuantificationUV260 QuantificationUV260 QuantificationUV260 QuantificationMALDIMALDI--TOF analysisTOF analysisHPLCHPLCHPLCHPLCFunctionalFunctional

QCQC--UV260UV260QCQC UV260UV260

99.5% Coupling Efficiency

80.0%

90.0%

100.0%

ial

20.0%

30.0%

40.0%

50.0%

60.0%

70.0%

Percent Full Length Mater

0.0%

10.0%

1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96 101

Length

P

QCQC--MALDIMALDI--TOFTOFQCQC MALDIMALDI TOFTOF

QCQC--HPLCHPLCQCQC HPLCHPLC

QCQC--FunctionalFunctionalQCQC FunctionalFunctional

QCQC--Interlude, why we don’t trust Interlude, why we don’t trust h i lh i lsearch engine resultssearch engine results

Liquid HandlingLiquid HandlingLiquid HandlingLiquid Handling

High ThroughputHigh ThroughputHigh ThroughputHigh Throughput

Why?Why?Why?Why?