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Oligonucleotide-Directed Mutagenesis

Trimer Phosphoramidites

In principle, the simplest approach for oligonucleotide-directed mutagenesis would be the use of trimer phosphoramidites. Of the 64 possible combinations of codons, only 20 codons would be required to cover the 20 amino acids, although, in practice, several codons will likely be duplicated depending on the organism. Our trimers use the protection scheme described1-3 by Kayushin et al. There is a concern that the sequence of the trimers has to be verified. For example, CAT coding for histidine, has to be differentiated from TAC, coding for tyrosine. These two trimers have virtually identical lipophilicity and their identity cannot be clearly confirmed by HPLC. This problem has been solved4 using HPLC electrospray mass spectrometric analysis of the trimers, which provides data confirming molecular weight and sequence.

In Table 1, the trimers, their coding amino acid and their reaction factor (RF) are listed. The reaction factor is critical since the trimers will likely be mixed and they have differing reactivity in the coupling reaction. RF for AAC is 1.0 and for TAC is 1.6. Therefore, 1.6 equivalents of TAC are needed for every 1.0 equivalent of AAC for equal coupling. Mixtures can easily be made using equimolar solutions or the molecular weight of each trimer has to be used to generate the appropriate weights of each trimer to use if mixing by weight. An example of the preparation of a mixture of all 20 trimers is shown in the right column of Table 1 and completed in the footnotes.

All of the trimers are now available individually so that researchers can prepare custom mixtures. A mixture of all 20 trimers designed to produce equal coupling of all 20 is also available. If you require custom production of a specific mixture, please e-mail support@glenresearch.com for a quotation and projected delivery.

TABLE 1: TRIMER CODING AND PHYSICAL PARAMETERS

Trimer Amino Acid abbreviation MW RF MWxRF mg/10µmol
(adjusted for RF)
AAA Lys K 1911.5 1.1 2102.65 21.0 (11)
AAC Asn N 1887.5 1.0 1887.50 18.9 (10)
ACT Thr T 1774.5 1.3 2306.85 23.1 (13)
ATC Ile I 1774.5 1.2 2129.40 21.3 (12)
ATG Met M 1780.5 1.3 2314.65 23.1 (13)
CAG Gln Q 1869.5 2.0 3739.00 37.4 (20)
CAT His H 1774.5 1.9 3371.55 33.7 (19)
CCG Pro P 1845.5 1.8 3321.90 33.2 (18)
CGT Arg R 1756.5 1.1 1932.15 19.3 (11)
CTG Leu L 1756.5 1.2 2107.80 21.1 (12)
GAA Glu E 1893.5 1.9 3597.65 36.0 (19)
GAC Asp D 1869.5 1.3 2430.35 24.3 (13)
GCT Ala A 1756.5 1.5 2634.75 26.3 (15)
GGT Gly G 1762.5 1.1 1938.75 19.4 (11)
GTT Val V 1667.5 1.9 3168.25 31.7 (19)
TAC Tyr Y 1774.5 1.6 2839.20 28.4 (16)
TCT Ser S 1661.4 1.3 2159.82 21.6 (13)
TGC Cys C 1756.5 1.5 2634.75 26.3 (15)
TGG Trp W 1762.5 2.4 4230.00 42.3 (24)
TTC Phe F 1661.4 1.3 2159.82 21.6 (13)
=530.0 mg

Example of Preparation of Trimer Mixture

Prepare 530 mg of the trimer mix, taking the amount (mg) for each trimer from the right column. Dissolve the trimer mix in dichloromethane (highest grade possible; acid-free). Evaporate to dryness to produce a homogenous mixture of all 20 trimers.

Example of Preparation of Trimer Mixture for the Synthesizer

Dissolve 530 mg, which is equivalent to 20X10 µmoles (normalized for RF) of the trimer mix in 2.0 mL of acetonitrile-dichloromethane mixture, 1:3 v/v to produce a 0.10N solution of trimers, ready for use in a synthesizer.

References

(1) A.L. Kayushin, M.D. Korosteleva, A.I. Miroshnikov, W. Kosch, D. Zubov, and N. Piel, Nucleic Acids Research, 1996, 24, 3748-3755.

(2) A. Kayushin, et al., Nucleos Nucleot, 1999, 18, 1531-1533.

(3) A. Kayushin, M. Korosteleva, and A. Miroshnikov, Nucleos Nucleot Nucleic Acids, 2000, 19, 1967-1976.

(4) T. Mauriala, S. Auriola, A. Azhayev, A. Kayushin, M. Korosteleva, and A. Miroshnikov, J Pharm Biomed Anal, 2004, 34, 199-206.

General Structure of Trimer Phosphoramidites,
where B=Abz, Cbz, Gibu, T

 
ItemCatalog No. PackPrice ($)
AAA Trimer Phosphoramidite 13-1000-95 50 µm 350.00
  13-1000-90 100 µm 700.00
AAC Trimer Phosphoramidite 13-1001-95 50 µm 350.00
  13-1001-90 100 µm 700.00
ACC Trimer Phosphoramidite 13-1011-95 50 µm 350.00
13-1011-90 100 µm 700.00
ACT Trimer Phosphoramidite 13-1013-95 50 µm 350.00
13-1013-90 100 µm 700.00
AGA Trimer Phosphoramidite 13-1020-95 50 µm 350.00
13-1020-90 100 µm 700.00
ATC Trimer Phosphoramidite 13-1031-95 50 µm 350.00
13-1031-90 100 µm 700.00
ATG Trimer Phosphoramidite 13-1032-95 50 µm 350.00
13-1032-90 100 µm 700.00
CAG Trimer Phosphoramidite 13-1102-95 50 µm 350.00
13-1102-90 100 µm 700.00
CAT Trimer Phosphoramidite 13-1103-95 50 µm 350.00
13-1103-90 100 µm 700.00
CCA Trimer Phosphoramidite 13-1110-95 50 µm 350.00
13-1110-90 100 µm 700.00
CCG Trimer Phosphoramidite 13-1112-95 50 µm 350.00
13-1112-90 100 µm 700.00
CGG Trimer Phosphoramidite 13-1122-95 50 µm 350.00
13-1122-90 100 µm 700.00
CGT Trimer Phosphoramidite 13-1123-95 50 µm 350.00
13-1123-90 100 µm 700.00
CTG Trimer Phosphoramidite 13-1132-95 50 µm 350.00
13-1132-90 100 µm 700.00
GAA Trimer Phosphoramidite 13-1200-95 50 µm 350.00
13-1200-90 100 µm 700.00
GAC Trimer Phosphoramidite 13-1201-95 50 µm 350.00
13-1201-90 100 µm 700.00
GAT Trimer Phosphoramidite 13-1203-95 50 µm 350.00
13-1203-90 100 µm 700.00
GCA Trimer Phosphoramidite 13-1210-95 50 µm 350.00
13-1210-90 100 µm 700.00
GCG Trimer Phosphoramidite 13-1212-95 50 µm 350.00
13-1212-90 100 µm 700.00
GCT Trimer Phosphoramidite 13-1213-95 50 µm 350.00
13-1213-90 100 µm 700.00
GGT Trimer Phosphoramidite 13-1223-95 50 µm 350.00
13-1223-90 100 µm 700.00
GTA Trimer Phosphoramidite 13-1230-95 50 µm 350.00
13-1230-90 100 µm 700.00
GTT Trimer Phosphoramidite 13-1233-95 50 µm 350.00
13-1233-90 100 µm 700.00
TAC Trimer Phosphoramidite 13-1301-95 50 µm 350.00
13-1301-90 100 µm 700.00
TCT Trimer Phosphoramidite 13-1313-95 50 µm 350.00
13-1313-90 100 µm 700.00
TGC Trimer Phosphoramidite 13-1321-95 50 µm 350.00
13-1321-90 100 µm 700.00
TGG Trimer Phosphoramidite 13-1322-95 50 µm 350.00
13-1322-90 100 µm 700.00
TTC Trimer Phosphoramidite 13-1331-95 50 µm 350.00
13-1331-90 100 µm 700.00
TTT Trimer Phosphoramidite 13-1333-95 50 µm 350.00
13-1333-90 100 µm 700.00
Trimer Phosphoramidite Mix 1 13-1991-95 50 µm 515.00
  (Mix of above 20 trimers) 13-1991-90 100 µm 1030.00
Trimer Phosphoramidite Mix 2 13-1992-95 50 µm 515.00
  (Mix of 19 Trimers - does not include TGC (Cys)) 13-1992-90 100 µm 1030.00

OTHER INSTRUMENT TYPES

All minor bases, RNA products and modifiers are packaged in septum-capped vials suitable for ABI and other instruments. If you would like another type of vial/column add the following to the end of the catalog number.

Monomers
For Instrument type
Add
Expedite
E
Beckman Oligo 1000
B
Pharmacia Gene Assembler
P
Mermade
M

Columns
For Instrument type
Add
Applied Biosystems 3900
A
Expedite
E
Mermade
M

Please inquire for availability
of columns for other
instrument types.

 

ATC Trimer

Antisense Trimer Phosphoramidites (Anticodons)

Trimer phosphoramidites have proven to be extremely valuable because they allow codon-based mutagenesis, which circumvents the common problems of codon-bias, frame-shift mutations, and the introduction of nonsense or stop codons. However, difficulties arise when trying to introduce mutations in multiple distal regions of a gene simultaneously. The synthesis of long oligonucleotides is required, which inevitably leads to lower sequence fidelity due to deletion mutants, depurination events and, to a lesser extent, mutations arising from deamination of cytidine, for example. An elegant solution to this problem is the use of Antisense Trimer Phosphoramidites. These trimers are the reverse complement of the cannonical ‘sense’ codons. When these antisense codons are put into the noncoding strand of a template DNA and amplified by PCR, they will code for the sense codon in the opposite strand of DNA. This allows the powerful technique of PCR Assembly to generate not only kilobase-sized genes from short 50mer oligonucleotides, but to simultaneously mutate multiple distal regions of that gene, as shown in the Figure below.

Figure : Simultaneous Mutation of Multiple Distal Regions of Gene

Figure 1

The sense and their corresponding antisense codons are listed in Table 1. Conveniently, many of our existing sense trimers can act as antisense codons. At present, we do not have enough data to accurately assign a Reaction Factor (RF) to the antisense trimers. Until those have been determined, a value of 1.425 is being used which is the average RF of the well-characterized sense trimers. As such, an antisense trimer mix designed to provide 20 amino acids represented equally would yield upon sequencing, some trimers that were over represented (i.e., >5%) and others under represented (<5%) upon sequencing. We will update the RF values when the data become available.

Figure : Simultaneous Mutation of Multiple Distal Regions of Gene

Sense codons

Antisense codons

MW

RF

5'->3'

5'->3'

 

(Temp)

AAA (Lys)

TTT

1572.4

1.425

AAC (Asn)

GTT

 

 

ACT (Thr)

GGT

 

 

ATC (Ile)

GAT

1780.5

1.425

ATG (Met)

CAT

 

 

CAG (Gln)

CTG

 

 

CAT (His)

ATG

 

 

CCG (Pro)

CGG

1851.5

1.425

CGT (Arg)

GCG

1851.5

1.425

CTG (Leu)

CAG

 

 

GAA (Glu)

TTC

 

 

GAC (Asp)

ATC

 

 

GCT (Ala)

TGC

 

 

GGT (Gly)

ACC

1863.5

1.425

GTT (Val)

AAC

 

 

TAC (Tyr)

GTA

1780.5

1.425

TCT (Ser)

AGA

1893.5

1.425

TGC (Cys)

GCA

1869.5

1.425

TGG (Trp)

CCA

1863.5

1.425

TTC (Phe)

GAA

 

 

ItemCatalog No. PackPrice ($)
ACC Trimer Phosphoramidite13-1011-9550 µm350.00
13-1011-90100 µm700.00
AGA Trimer Phosphoramidite13-1020-9550 µm350.00
13-1020-90100 µm700.00
CCA Trimer Phosphoramidite13-1110-9550 µm350.00
13-1110-90100 µm700.00
CGG Trimer Phosphoramidite13-1122-9550 µm 350.00
13-1122-90100 µm700.00
GAT Trimer Phosphoramidite13-1203-9550 µm 350.00
13-1203-90100 µm700.00
GCA Trimer Phosphoramidite13-1210-9550 µm350.00
13-1210-90100 µm700.00
GCG Trimer Phosphoramidite13-1212-95 50 µm350.00
13-1212-90100 µm700.00
GTA Trimer Phosphoramidite13-1230-9550 µm350.00
13-1230-90100 µm700.00
TTT Trimer Phosphoramidite13-1333-9550 µm350.00
13-1333-90100 µm700.00

http://www.glenres.com/Catalog/odm.html

 
Please contact Glen Research if you have any questions or comments!
 
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