The oligonucleotide synthesis on the solid support is a complex, multistep process. The production takes place in 3 ', 5'-direction and requires four steps for the chain extension to a base:

1. Removal of the 5'-DMT-protective group (deblocking)

2. chain extension by coupling a Amidites (Coupling)

3. acetylation of the 5'-OH groups, which have not reacted (Capping)

4. Oxidation of the phosphite intermediate to phosphate triester (Oxidizing)

Although the individual steps proceed with very good yields, but not quantitatively! The coupling yield is dependent on the sequence and the length, wherein the base G at the worst and the coupling yields are poor by steric hindrance from a chain length of about 50 bases. Adequate ongoing synthesis machines using quality chemicals coupling yields of 98 to be greater than 99% achieved depending on the length and sequence.

The table below shows that the total yield depends strongly on the average coupling rate and makes it clear that even with short oligonucleotides (20 mer) and good coupling rate (99%) only 83% full length can be achieved.

length

Full length Yield [%]

98% coupling rate

98.5% coupling rate

99% coupling rate

20mer

67

75

83

40mer

45

55

68

60mer

30

41

55

80mer

20

30

45

100mer

13

22

37

After the synthesis the crude product containing the following impurities:

Base protecting groups and salts

Thus, the structure of the internucleotide bond is selective, the heterocyclic bases must be protected. Classic protecting groups are benzoyl for adenosine and cytidine and isobutyryl for guanosine. After completion of the synthesis, a cleavage with ammonia. The amides formed in this process are removed by precipitation of the oligonucleotide.

Truncated sequences without DMT group

The so-called trityl-off truncated sequences arising from incomplete couplings and by post synthetic strand break. For optimal running standard syntheses (20 mer, coupling yield 99%) of the share amounts to about 20%.

The post-synthetic strand break occurs when the ammonia treatment at all positions where the heterocyclic base has been removed. The loss of the base is carried out under acidic conditions in the deblocking mainly for adenosine and plays a role in long oligonucleotides. The trityl-off truncated sequences can be distinguished removed by HPLC purification.

Truncated sequences with DMT group

The so-called trityl-on truncated sequences arise because the activities carried out for chain extension reactions not proceed quantitatively and postsynthetic strand breaks at sites where the heterocyclic base missing (depurination). The complete separation is possible only by PAGE.

. The company offers the following PURIMEX purifications:

desalination

Desalting by ethanol completely removes all base protecting groups and salts. Shorter failure sequences (<10 bases) remain largely in the supernatant. From a length of 35 bases, HPLC purification is required. The price of desalination is included in the base price!

The solid delivery volumes amounted to 2 nmol (10 nmol scale), 10 nmol (50 nmol scale), 50 nmol (200 nmol scale) and 200 nmol (1000 nmol scale)

Easy HPLC Purification

The trityl-on HPLC purification completely removes all truncated sequences containing no trityl group. In addition, a separation of salts and base protecting groups. After cleavage of the trityl group used "handle" the product is isolated by ethanol precipitation and dissolved in water. Subsequently, the yield is determined by measuring the UV absorbance at 260 nm is determined and the oligonucleotide adjusted by adding water to a concentration of 100 uM. From a length of 35 bases, this purification is performed automastisch.

The solid delivery volumes are 1.2 nmol (10 nmol scale), 6 nmol (50 nmol scale)

30 nmol (200 nmol scale) and 120 nmol (1000 nmol scale).

HPLC purification of RNA we do not offer, because the separation of truncated sequences succeeds only moderately and the process is difficult to reproduce.

designation

Price [euro] / purification

10 nmol

50 nmol

200 nmol

1000 nmol

Easy HPLC Purification

7

8th

10

15

Double HPLC Purification

Following the trityl-on HPLC purification after cleavage of the trityl group by a second HPLC-purification. In this way, oligonucleotides having base modifications and Trityl-On containing truncated sequences, for example, be caused by strand breaks after synthesis, largely removed.

Oligonucleotides that have an amino or thiol linker, are at least twice purified HPLC

The solid delivery volumes amounted to 1 nmol (10 nmol scale), 5 nmol (50 nmol scale)

25 nmol (200 nmol scale) and 100 nmol (1000 nmol scale).

designation

Price [euro] / purification

10 nmol

50 nmol

200 nmol

1000 nmol

Double HPLC Purification

14

15

20

30

Triple HPLC Purification

In the post-synthetic introduction of dyes via amino-oligo / NHS ester or thiol-oligo / maleimide, the product is purified three times in total HPLC. With the first HPLC (TRON) is carried out a separation of the duly amino-thiol linkers or linker full length product from the condensed sequences containing no trityl group. After removal of the trityl group, a reaction that is reversible unfortunately, there is a second HPLC (dripped) in order to remove trityl On-containing sequences. After isolation of the product is reacted with the dye, and then separated with the third HPLC unlabeled oligonucleotide and excess dye.

The solid delivery volumes are 4 nmol (50 nmol scale), 20 nmol (200 nmol scale) and 80 nmol (1000 nmol scale).

designation

Price [euro] / purification

50 nmol

200 nmol

1000 nmol

Triple HPLC Purification

24

30

45

HPLC purification fourfold

Four times the HPLC purification is required for dual-labeled oligonucleotides, when both an amino linker, a thiol linker as well be used. In the 1st HPLC isolation of the Vollängenoligos (1x 1x on aminolinker, the thiol linker) is done that includes 2 trityl. After removal of the amino linker-trityl a 2nd time Tron is purified. Subsequently, the trityl group is removed in a thiol linker and the dripping product in the 3rd HPLC isolated in a special process. After implementation of the oligonucleotide with the two dyes not labeled with the 4th HPLC oligonucleotide and excess dyes is separated.

The solid delivery volumes are 3.5 nmol (50 nmol scale), 17.5 nmol (200 nmol scale) and 70 nmol (1000 nmol scale).

designation

Price [euro] / purification

50 nmol

200 nmol

1000 nmol

HPLC purification fourfold

32

40

60

HPLC purification fivefold

If the dual-labeled probe should be particularly clean, takes place after the first dye marking the 4th HPLC. After the product has been implemented with the second dye, the separation of excess dye and single-labeled oligo in a 5. HPLC.

The solid delivery volumes are 3 nmol (50 nmol scale), 15 nmol (200 nmol scale) and 60 nmol (1000 nmol scale).

designation

Price [euro] / purification

50 nmol

200 nmol

1000 nmol

5x HPLC Purification

40

50

75

Polyacrylamidgelelektophorese (PAGE) purifying DNA / RNA

The PAGE purification under denaturing conditions is the most appropriate method for obtaining highly purified oligonucleotides, particularly in combination with HPLC. The separation is effected by charge and mass and globular structure. After synthesis and deprotection is at first a DNA double HPLC (TRON / troff, not included) performed.Following the PAGE purification is carried out.

RNA are available exclusively from PAGE purified, since the proportion of full-length product compared to DNA, is due much lower by significantly poorer coupling yields. An HPLC we do not offer quality reasons. The crude product is purified after deprotection directly over the gel. After isolation of the full length product a Sep-Pak® is (not included) performed desalination. When RNA is to be marked with dyes postsynthetically, a 2nd SepPak desalination is required.

Purification of oligonucleotides with PAGE leads to high purity products.

The solid delivery amounts up to a chain length of 50 bases are 2 nmol (50 nmol scale), 10 nmol (200 nmol scale) and 50 nmol (1000 nmol scale).

designation

Price [EUR]

PAGE purification DNA

100

PAGE purification RNA

100

Sep-Pak ® desalination DNA / RNA

After PAGE purification, the products are desalted on a cartridge. With dye labels of RNA another SepPak desalination is required

designation

Price [EUR]

Sep-Pak ® desalination DNA

20:00

Sep-Pak ® desalination RNA

30,00

Double Sep-Pak ® desalination RNA

60,00

Sterile filtration

Oligonucleotides that are used in cell culture, are dissolved after precipitation in high purity, DEPC treated water and filtered directly into the packaging unit through a 0.2 micron membrane.

designation

Price [EUR]

Sterile filtration

10,00

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