Post on 06-Jan-2017
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?