Frequently asked questions: Custom Oligonucleotide Synthesis

FAQ_Oligonucleotides_080827.doc June 2005 1 of 9

Frequently asked questions: Custom Oligonucleotide Synthesis A) Oligonucleotide synthesis 1. What kind of chemistry does Thermo Scientific us e?

Thermo Scientific routinely uses solid phase synthesis and performs phosphoramidite chemistry.

2. How is solid phase synthesis done? Synthesis is performed from the 3’-end to the 5’-end of the oligonucleotide (opposite to direction in nature). The synthesis cycle comprises: a) Loading of solid support phase (CPG resin), where 3’-terminal phosphoramidite (PA) is attached. This PA is protected at its 5’-OH in order to prevent unwanted reactions. b) Deprotection of 5’-OH of support-bound PA c) Coupling of next PA (addition of next base), formation of phosphite intermediate d) Capping reaction in order to inactivate all non-reactive PA molecules e) Oxidation of phosphite to phosphate (stabilisation of linkage) f) Re-start of cycle at b) or cleavage of full-length oligonucleotide from solid support at the end of synthesis

3. What is the longest oligonucleotide that Thermo Scientific synthesise?

Thermo Scientific is capable to synthesise oligonucleotides up to a length of 140 bases. Are there other length restrictions? Yes, not every oligo (especially modified ones) can be synthesised up to our maximum length. There are limitations in the synthesis chemistry (p.e. some labile dyes cannot cope with the aggressive chemicals for long time) and solid-support material (pore size limits the maximum oligo length). The following table shows some general limits:

oligo type maximum length

in bases unmodified 140 5’-modified* 100 *except for: 5’-HRP 35 3’-modified** 80 **except for: 3’-Thiolink C3, 3’-inverted end, 3’-Cy3 and 3’Cy5

45

double-labelled oligos 40 RNA 50 2’-O-Methyl-RNA 105

other lengths on request!

deprotection

Tr off

addition ofnext

molecule

oxidation

repeat

capping

2 %

1.6 %0.4 %

cleavage


4. What does the OD 260nm value stand for? The OD260nm (Optical Density) value denotes the amount of oligonucleotide you have received. The conversion of OD260nm values to nanomoles depends on the molecular weight of the individual oligo. Therefore no general applicable conversions can be offered.

equals or* 30 µg ss-DNA 4.5 nmol of a 20mer 40 µg ss-RNA 1.8 nmol of a 50mer

1 OD260nm

50 µg ds-DNA 0.9 nmol of a 100mer * only valid for ss-oligos with equal base composition of all four bases.

5. Why does Thermo Scientific sell oligos in OD amo unts rather than nmol?

The reason is that the same amount of reagents is needed in order to produce 1 OD260nm of an oligonucleotide – whether it is a 20mer or a 100mer. In contrast to this, we would need 5 times the reagents for 1 nmol of a 100mer than for a 20mer. So in order to keep our base prices independent from the actual oligo length, we have to sell “length-independent” scales.

What does the synthesis scale tell me then? The synthesis scale represents the amount of starting material in µmol at the beginning of synthesis (=loading amount of synthesis column). It is no indication for product yield to be expected! Yields of full-length oligonucleotide are lower than the starting amount due to various reasons: - the coupling efficiency of different bases or modifications is not 100%. (For example, the standard coupling efficiency of the 4 “normal” DNA bases is about 98-99,5%.) Coupling of modifications can be as low as 50%. Coupling efficiency is influenced by oligo length, oligo sequence and types of modifications. - Thermo Scientific purifies all oligonucleotides by RP-HPLC. Thus, the yield decreases again, because a majority of unwanted by-products is removed from the crude oligonucleotide. Please refer to the following tables for information on oligo yields in OD (Optical Density): DNA oligonucleotides – Yields in OD oligo length: 16-40 mer (guaranteed for a 20 mer) synthesis scale purification oligo type primer 0.02 0.04 0.2 1.0 10

unmodified 2 2 4 10 30 300 single modification --- 1 2 5 15 150

HPLC

double modification --- 0,5 1 2,5 7,5 75 unmodified --- 0,5 1 3 10 100 single modification --- 0,3 0,5 1,5 5 50

PAGE

double modification --- 0,2 0,3 0,5 2,5 25 oligo length: 8-15 or 41-100 mer purification oligo type primer* 0.02 0.04 0.2 1.0 10

unmodified 2 1,5 3 7 25 250 single modification --- 0,7 1,5 3 12 120

HPLC

double modification* --- 0,3 0,7 2 6 60 unmodified --- 0,3 0,7 2,5 7 70 single modification --- 0,2 0,3 1 4 40

PAGE

double modification* --- 0,2 0,2 0,3 2 20 * not available for oligos > 40 bases

oligo length: >100 mer synthesis scale purification oligo type 0.02 0.04 0.2 1.0 10

unmodified 1 2 5 15 150 HPLC single modification 0,5 1 2,5 7,5 75 unmodified 0,3 0,5 1,5 5 50 PAGE single modification 0,2 0,3 0,7 2,5 25

For other products, please refer to our current pr icelist.


6. How are Thermo Scientific oligonucleotides quant ified?

Oligonucleotides are quantified by measuring their UV optical density (OD) at a wavelength of 260 nm. The amount of oligonucleotide is determined from its base composition and the measured OD value with the following formula (formula 1):

Formula 1

)*92.0*75.0*17.1*54.1(

][*100][

TCGA

ODnnmoln

+++=

n [OD]: OD value n [nmol]: amount in nmol A,G,C,T: number of the respective bases in the oligo 1.54; 1,17; 0,75;0,92: extinction coefficients for each type of base

Multiplying this value by the molecular weight of the oligo reveals the amount in nanogram (formula 2). 1 OD equals approximately 33 µg ss-DNA (equimolar mixed sequence of all 4 bases). Division by the volume used for dissolving displays the concentration (formula 3):

Formula 2 Formula 3

1000

]/[*][]µ[

molgMWnmolngm =

]µ[

][*1000]µ/[

lv

nmolnclpmolc ==

m [µg]: amount in µg n [nmol]: amount in nmol MW [g/mol]: molecular weight (see certificate) c [pmol/µl]: concentration v [µl]: volume of oligo solution

7. How can I requantify my oligonucleotide? Oligonucleotides can be easily quantified by using a UV photometer and a quartz cuvette. If not already dissolved, resuspend your oligonucleotide in 400 µl of sterile water. Take a 10 µl aliquot, dilute it with 990 µl of water and measure its extinction at 260 nm. The reading should be typically between 0.1 and 1.5 units. After multiplication with the dilution factor of 40 (400 µl / 10 µl) you get the amount in OD of the entire oligo sample. You can use formula 1 (see above) to calculate the amount in nmol. To determine the concentration of your oligo solution you divide this amount with the solvent volume used.

8. How does Thermo Scientific calculate the molecul ar weight (MW) of an oligonucleotide? The molecular weight MW of your oligo is calculated from the base composition and its modifications (formula 4). This formula is used for calculation of the amount in µg and as a reference value for MALDI –TOF analysis. Formula 4

[ ]62*2.304*2.289*2.329*2.313]/[ mod −++++= MWTCGAmolgMWoligo

A,G,C,T: number of the respective bases in the oligonucleotide 313.2; 329.2; 289.2; 304.2: molecular weight for each type of base

MWmod [g/mol]: molecular weight of an modification (if present) Please note that it is not possible to calculate mo lecular weights for oligos with mixed bases as such oligos represent a mixture of differe nt individual oligos!


9. How does Thermo Scientific calculate the melting temperature (Tm)? The melting temperature Tm of an oligo is the temperature at which 50 % of an oligonucleotide and its perfect complement are in duplex. This parameter is essential for all applications in molecular biology where hybridisation of DNA strands is involved. Typically, annealing or hybridisation are performed at 5 °C below the Tm of a duplex. DNA sequence, length and concentration, salt concentration and the presence of formamide or DMSO are the main factors in influencing Tm. Please note that it is not possible to calculate me lting temperatures for oligos with mixed bases as such oligos represent a mixture of differe nt individual oligos! For oligos longer than 15 bases, Thermo Scientific use the „Nearest Neighbour“ method (Rychlik et

al, Nucl Acid Res 1990, 18, 6409-6413) to calculate Tm (formula 5). This method is considered to be one of the more accurate calculations of Tm for a wide range of oligonucleotide lengths. It takes into account each pair of neighbouring bases in a sequence, as well as oligonucleotide and salt concentration:

Formula 5

)(log*6.1615.273)4/ln(*

*1000 ++−

+∆+∆= Kc

CRSA

HTm

∆H [kcal/mol]: sum of the nearest neighbour enthalpy changes ∆S [cal/mol]: sum of the nearest neighbour entropy changes A = -10,8 cal: constant entropy factor for helix initiation R = 1.98 cal/(°C*mol): universal gas constant C = 250 pmol/l: concentration of oligonucleotide c(K+) = 50 mmol/l: concentration of monovalent cations

For oligos shorter than 15 bases Thermo Scientific use the simple but therefore more accurate „Wallace rule“ (formula 6). This equation was developed for hybridising short oligonucleotides to membrane-bound DNA. Formula 6

)(*C4)(*C2 CGTATd +°++°=

Td: temperature at which 50 % of an oligo and its perfect surface-bound complement are in duplex

(salt concentration 0.9 M) A,G,C,T: number of the respective bases in the oligo

To use this equation for solution based experiments you should add 8 °C to the result. All current formulae do not take into account any type of modification such as dyes, haptens or anchor groups. These modifications tend to decrease the Tm value (internal modifications have higher grade of influence than terminal ones). If your browser allows JavaScript, you can calculate the Tm value of your oligos using our Physical parameter calculator on our online ordering page www.thermo.com/oligos.

10. Does my oligonucleotide have a phosphate on the 5ór 3´ end? No, it doesn’t. Unless ordered otherwise, all our custom oligonucleotides are synthesised with free hydroxyl groups on both ends (3ánd 5´). However, if required we can synthesise your oligo with 5ánd/or 3´phosphate (phosphorylation).

11. Does Thermo Scientific offer annealing of two o ligonucleotide strands? Yes, we offer the service of hybridisation of two DNA or RNA strands. Please order each complementary strand in 5´- to 3´- direction and indicate the hybridisation in the annotations.


B) Handling and storage 1. How are the oligonucleotides supplied?

Unless ordered otherwise, Thermo Scientific oligonucleotides are supplied lyophilised and ready-to-use in single tubes. Concentration adjustment, delivery in 96-deepwell plates and additional aliquots are available on request.

2. What are the recommended storage conditions for oligonucleotides? Our oligonucleotides are shipped in dried state, unless ordered otherwise. Thus, they are more stable and can be stored at temperatures below 4°C for at least 1 year. After dissolving, oligonucleotide solutions have to be stored at temperatures below –20 °C and stay stable for some months in general. Repeated freeze-thaw cycles have to be avoided to prevent untimely degradation and contamination of stock solutions. Oligonucleotides with dye modifications have to be protected from light at all times to prevent bleaching of the dye.

3. How should oligonucleotides be dissolved?

To dissolve your oligonucleotides we recommend nuclease-free, sterile water (pH ~ 7), or sterile TE buffer (pH 7,5 ~ 8). Upon receipt, please spin down the oligonucleotide tubes, as part of the product might stick to the lid. The volume to prepare a 100 µM (= 100 pmol/µl) oligonucleotide solution can be found in the quality certificate that accompanies every oligonucleotide delivery. In general, oligonucleotides will dissolve in this volume - when vortexed - within a few minutes. If a standard oligonucleotide is reluctant to dissolve, careful warming of the solution for 10-30 minutes up to 37°C is recommended.

C) Quality and Purification 1. What types of purification does Thermo Scientifi c offer?

Thermo Scientific systematically purify all oligonucleotides by reverse-phase HPLC (High Pressure Liquid Chromatography) as standard at no extra cost. PAGE purification (Poly-Acrylamide-Gel-Electrophoresis) can be ordered as further option. We strongly recommend PAGE purification for oligonucleotides longer than 50 bases, that are used for cloning, mutagenesis or gene synthesis experiments. For special purity requirements Thermo Scientific offers Ion Exchange Chromatography (IEC), too.

2. What kind of quality control does Thermo Scienti fic offer? We perform QC on our products and on all our instruments to ensure that you only receive products of the highest quality. Our routine QC procedure for oligonucleotides covers a daily control of all synthesising machines, where product samples are taken randomly and analysed by MALDI-TOF Mass Spectrometry and Capillary Electrophoresis (CE). If required, these services can also be ordered by our customers.


3. What does the HPLC-Graph on the certificate show ? The graph shows the absorption data (detection at λ = 260 nm) collected during the HPLC purification run of your oligonucleotide. It is not the analysis of the purified product!

Unmodified oligonucleotides or oligos with phosphate, biotin, amino-MMT or thiol modification display the following type of HPLC profile: At first, capped sequences (by-products during synthesis) and protection groups are being eluted. The full-length product peak usually appears after 1.5 – 2 minutes. The black mark in above profile denotes the part of the product peak, that is being collected during HPLC purification. Shorter products (so-called n-x products) usually follow the main product in the “tail” of the peak. These aren’t collected. Oligos with fluorescent or DIG-label can display the following type of HPLC profile: The black mark denotes the fraction of the crude product that is being collected. If several dye-labelled peaks appear, they are collected separately and are analysed by MALDI-TOF Mass Spectrometry to identify the fraction that includes the desired product with the correct molecular weight.

unmodified oligo

oligo with degraded dye

dye - labelled oligo desired product

full - length product (DMT on )

n-x products (DMT on )

protection groups

capped sequences (DMT off )


D) Product offering 1. What kind of modifications are available?

Thermo Scientific offers one of the broadest ranges of modifications for DNA oligos - such as: • Functionalisation with: amino, phosphate or thiol groups • Fluorescent dyes, DIG, HRP, psoralene, dinitrophenyl (DNP) or cholesteryl labelling.

Our table (fluorescent dyes) with all available fluorescent dyes sorted by their emission and excitation values helps you to find the right dye for your equipment

• Double-labelled probes for Real-time PCR • LNA™ (locked nucleic acid) • RNA, 2’-O-Methyl-RNA • Unnatural bases, e.g. Br-dU, I-dU, F-dU, 5-Methyl-dC, N6-Methyl-dA, N4-ethyl-dC,

Nitroindole, Aminopurine, dP, dK, Inosine • Phosphothioates (PTOs) • Spacers of different lengths • Doubler molecules for branched oligonucleotides • Wobble bases (degenerate oligonucleotides) • And many other special modifications The standard collection of modifications is shown in our price list. A broad spectrum of special modifications is available on request by e-mail to [email protected]

2. Does Thermo Scientific offer GMP oligos? No, we don’t offer “real” GMP oligos, but customers with enhanced quality needs for diagnostics or resale purposes can choose our I.M.P. (Interactiva Manufacturing Procedure) oligo option. For only little extra costs the I.M.P. option includes: - handling of your order separately from routine production by specially trained staff - production of your oligos on special synthesisers with documented reagent lots - full documentation of all production steps - special purification procedure for highest product purity - documented MS-analysis of every oligonucleotide - suppliers audit can be done by our customers If you are interested in the I.M.P. option, please contact [email protected]


3. What modification is used in which application? Please refer to the following table:

Application

PC

R

Rea

l-tim

e P

CR

FR

ET

Seq

uenc

ing

Fra

gmen

t Ana

lysi

s

Clo

ning

/ Li

gatio

n

Mut

agen

esis

Gen

e sy

nthe

sis

Hyb

ridis

atio

n

Mic

roar

rays

Ant

isen

ce a

ssay

s

Str

uctu

re-r

elat

ions

hip

assa

ys

Bio

logi

cal

func

tiona

lisat

ion

assa

ys

Imm

obili

satio

n on

sol

id

surf

aces

Cou

plin

g of

add

ition

al

mod

ifica

tions

Gen

e si

lenc

ing

Enhanced purification PAGE x x x

Functional Groups NH2 (Aminolink) x x x SH (Thiollink) x x x

PO4 (Phosphate) x x Biotin x x x DIG (Digoxigenin) x x HRP (Horseradish Peroxidase)

x x

Fluorescence Dyes Fluorescent dyes x x x x x x Double-labelled fluorescent probes

x x x

Dye-dT x x x x x Dabcyl x x x x

Internal Modifications PTO (Phosphothioate-Oligo)

x x x

Methyl-Phosphonate-Oligo x x

2´ O-Methyl-RNA x x 2´deoxy Inosine x x x

2´deoxy Uridin x Biotin-dT x x x x X Amino-dT x x Dye-dT x x x dSpacer x x halogenated bases (5-Br-dC, 5´Br-dU, 5-I-dC, 5-I-dU)

x x

Special Modifications Carbon Chain Spacers x x Ethylenglycol Spacer x x 3´Block (C3-Spacer) x x x x Inverted End (3´-3-linkage)

x x x

Asymmetric doubler x x Symmetric doubler x x

RNA RNA x x x

si-RNA x


4. Can I combine every single modification with ano ther one? No, due to the different types of synthesis strategies some combinations of our modifications are not possible. Please refer to the following table to find out if your desired combination of standard modifications for DNA oligos is available. Combinations marked in red are not available!

3‘-terminal

5‘-terminal Am

inol

ink

C6

Thi

olin

k C

3

Pho

spha

te

Bio

tin

Dig

oxig

enin

Dab

cyl

Bla

ck H

ole

Que

nche

r

Inve

rted

end

3‘-B

lock

(C

3 S

pace

r)

Flu

ores

cein

6-F

AM

TA

MR

A

JOE

RO

X

Cy™

dye

s

AM

CA

Bod

ipy™

dye

s

Mar

ina

Blu

e

Pac

ific

Blu

e

Ore

gon

Gre

ens

Rho

dam

ine

Gre

en

Rho

dam

ine

Red

Tex

as R

ed

Aminolink C6

Thiolink C6

Phosphate

Biotin

Digoxigenin

Fluorescein

6-FAM

HEX

TET

TAMRA

JOE

ROX

Cy™dyes

IR™dyes

AMCA

Bodipy™ dyes

Marina Blue

Pacific Blue

Oregon Greens

Rhodamine Green

Rhodamine Red

Texas Red

HRP

5. On which end (3ór 5´) of the oligonucleotide sh ould modifications be attached?

Most of our modifications are available for both ends. You can choose the side that your application recommends. The coupling possibilities for each modification are indicated in our price list. If your assay procedure does not otherwise require, Thermo Scientific recommends to attach modifications to the 5´-end of your oligonucleotide (p.e. most PCR applications are still possible if the modification is attached to the 5'- end). In contrast, modifications at the 3'- end usually block this end for further enzymatic reactions (nevertheless, sensitive applications might detect elongation even of 3'-modified oligonucleotides. For complete blocking we recommend inverted end or C3-Spacer modification).

6. What does Thermo Scientific charge for wobble ba ses (degenerate primers)?

Standard wobbles (equimolar ratio of the respective bases) are offered free of charge – even at the 3’-end of your oligo. If you need a special mixture (p.e.: 70% A and 30% G), a special handling fee for individual mixtures applies (price list). Please order degenerate primers using the official IUB-code (please refer to table on our web site. An oligo with wobbles represents a mixture of many different oligonucleotides. Thus, no molecular weight or Tm value can be calculated for these. Please note: when using degenerate oligos for PCR, please keep in mind that the 100% homologue to your template is only a small fraction of the total oligo mixture – so an increase of primer concentration might be necessary to obtain results.

Documents

Frequently asked questions: Custom Oligonucleotide Synthesis