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WO2009097673A1 - Synthèse d’adn monobrin - Google Patents

Synthèse d’adn monobrin Download PDF

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Publication number
WO2009097673A1
WO2009097673A1 PCT/CA2008/000224 CA2008000224W WO2009097673A1 WO 2009097673 A1 WO2009097673 A1 WO 2009097673A1 CA 2008000224 W CA2008000224 W CA 2008000224W WO 2009097673 A1 WO2009097673 A1 WO 2009097673A1
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Prior art keywords
molecule
template
target
target molecule
bases
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Thuraiayah Vinayagamoorthy
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Bio ID Diagnostic Inc
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Bio ID Diagnostic Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Definitions

  • This invention relates to methods of synthesis of single-stranded DNA or like polynucleotides of desired nucleotide sequence. More particularly, the invention relates to methods of synthesis of long stretches of single-stranded DNA of increased length and/or molecular weight.
  • Short lengths of single-stranded DNA are used in a number of techniques employed in molecular biology, for example as primers employed in DNA amplification using the polymerase or ligase chain reaction (PCR or LCR), as primers used for DNA sequencing, or as labeled probes used for sequence identification in samples of DNA.
  • Such short lengths of single-stranded DNA are normally produced by attaching a base nucleotide to a solid support and then linking one nucleotide at a time to the growing chain to create a nucleotide polymer of predetermined sequence. This is generally carried out within a commercial nucleic acid synthesizer.
  • sequences up to 100 bases in length may be synthesized with some difficulty, but sequences of more than 80 bases are not normally produced in this way. Indeed, sequences of only 20-35 base pairs are most commonly produced using such techniques. For short primers required for PCR or DNA sequencing, this limitation on sequence length is not usually a problem. However, other techniques may require sequences of 100 bases or more, e.g. those designed to introduce restriction sites into DNA molecules, those used in a multi-loci gene sequencing technique described in U.S. patent 6,197,510 issued to T. Vinayagamoorthy on March 6, 2001, or those involving the synthesis of genes or for use in making microcircuits by means of nanotechnologies.
  • a template molecule to anneal to one or more parts of a larger target molecule while the target molecule is constructed.
  • the template molecule may then be removed by digestion.
  • Both the template molecule and the one or more parts of the target molecule are short enough to be synthesized by conventional techniques (e.g. they are of 100 bases or less, preferably 80 bases or less and, more preferably, 60 bases or less). Nevertheless, a target molecule of larger size can be assembled by ligation or constructed polymerization from the smaller part or parts.
  • a digestion enzyme specific to uracil e.g. Uracil-N-glycosylase (UNG) may then be used to remove the template, thus freeing the target DNA molecule (which, like all DNA, contains no uracil).
  • an exemplary embodiment of the present invention provides a method of producing single-stranded DNA.
  • the method comprises providing an oligonucleotide template molecule having a sequence that is complementary to a part of a target single- stranded DNA molecule of length greater than the template molecule, the template oligonucleotide incorporating one or more uracil bases; providing at least one part of the target molecule including a base sequence complementary to a part of the template molecule; annealing the at least one part of the target molecule to the template molecule and forming the complete target molecule by a method selected from the group consisting of ligating at least two adjacent parts of the target molecule while annealed to the template molecule, extending at least one part of the target molecule to form a sequence complementary to a remainder of the template molecule by nucleotide polymerization, and a combination of both the ligating and extending methods; and separating the template molecule from the target molecule.
  • the separating of the template molecule from said target molecule is preferably carried out by digesting the template molecule with a digestion enzyme specific to molecules containing uracil bases, .e.g. uracil-N-glucocylase (the enzyme chemically cuts the template into fragments).
  • a digestion enzyme specific to molecules containing uracil bases .e.g. uracil-N-glucocylase (the enzyme chemically cuts the template into fragments).
  • other techniques may be employed, e.g. by employing heat and/or alkali to pH 12.0, but are often less desirable.
  • the enzymatic method does not have to be followed by filtration, although this is normally done for greater purity and for downstream purposes.
  • Techniques involving separation by heat or alkali have to be followed by filtration under conditions favoring separation to prevent re- annealing of the template and the target.
  • the target molecule part(s) and the template each has a length smaller than 100 bases, more preferably smaller than 80 bases,
  • the target molecule is isolated from other reaction products and starting materials by a suitable method, e.g. by membrane filtration.
  • Another exemplary embodiment of the invention provides a method of producing a single-stranded polynucleotide molecule, which comprises: providing a template polynucleotide molecule having a sequence of bases complementary to a target polynucleotide molecule, the template sequence including at least one uracil base; annealing to the template molecule an intermediate polynucleotide molecule having a sequence complementary to the template molecule; cutting (nicking) the intermediate polynucleotide molecule into two fragments while annealed to the template molecule and extending one of the fragments to replace the other by nucleotide polymerization employing nucleotide monomers of increased molecular weight or ionic charge compared to nucleotide monomer residues forming the intermediate molecule, thereby forming the target polynucleotide molecule having sequence complementary to the template; and separating the template from the target polynucleotide molecule, thereby forming a single- stranded target
  • This exemplary embodiment can be used to prepare single-stranded DNA sequences that may be of a length that can be produced economically by conventional techniques, but includes a fragment that is of increased molecular weight or ionic charge compared to "natural" DNA, i.e. DNA that incorporates dNTPs found in natural DNA.
  • the resulting single-stranded DNA may be used, for example, as probes used for PCR or DNA sequencing that creates fragments of increased molecular weight or ionic charge compared to fragments of similar numbers of base pairs, thereby enabling improved separation from such fragments using gel electrophoresis or the like.
  • a method of producing a single-stranded polynucleotide molecule which comprises: providing a template polynucleotide molecule comprising at least one uracil base and having a sequence that is complementary to at least a part of a target polynucleotide molecule; providing an additional polynucleotide molecule having a sequence corresponding to at least a part of the target molecule; allowing the template molecule and the additional molecule to anneal together; modifying the additional molecule while annealed to the template to form the target molecule; separating the target molecule from the template by providing an enzyme that digests polynucleotides containing uracil bases, thereby freeing the target molecule as a single-stranded polynucleotide; and isolating the single-stranded target molecule from remaining reaction products and starting materials.
  • Fig. 1 is a diagram illustrating, in simple form, one possible method according to the present invention
  • Fig. 2 is a diagram illustrating, in simple form, a second possible method according to the present invention.
  • Fig. 3 is a diagram illustrating, in simple form, an alternative embodiment of the invention.
  • Fig. 4 is another diagram illustrating, in simple form, an alternative embodiment of the invention.
  • DNA is a polymer comprising a chemically-linked chain of nucleotide monomers, each of which consists of a sugar (deoxyribose), a phosphate and one of four kinds of nucleobases ("bases”), namely adenine (A), thymine (T), cytosine (C), and guanine (G).
  • bases adenine (A), thymine (T), cytosine (C), and guanine (G).
  • a DNA molecule is able to anneal (i.e. reversibly join to) a second strand of DNA having a complementary sequence of bases, that is to say, a sequence where there is a T on one strand aligned with an A on the other, and a G on one strand aligned with a C on the other.
  • the base uracil (U) is also complementary to A, so a molecule having one or more of its T bases replaced by U will anneal to a complementary strand having an aligned A for every U.
  • the base uracil does not normally occur in DNA (it is a component of RNA), so synthesized DNA target molecules normally do not contain U.
  • a single-stranded DNA molecule of greater length e.g. 100 nucleotides or more
  • a target DNA molecule i.e. a molecule that is desired to be produced
  • a template molecule is first constructed. This is a polynucleotide of a length that can be synthesized by conventional techniques and having a sequence that is complementary to a part of the intended sequence of the target DNA molecule, but including at least one U instead of a T that would normally be present. The reason for this substitution will be apparent from the description below.
  • At least one short part of the target molecule is then also synthesized using a conventional technique, and the template molecule and target part(s) are allowed to anneal.
  • the completion of the construction of the target molecule then follows by one of at least two procedures.
  • the two procedures are illustrated in simple form in Figs. 1 and 2 of the accompanying drawings.
  • Fig. 1 illustrates a polymerization technique
  • Fig. 2 illustrates a ligation technique.
  • Step A represents the provision of both one part 10 of a target molecule and a template molecule 11.
  • the method of synthesizing the target part and template may be carried out conventionally, for example, as disclosed in Molecular Cloning:
  • the target part 10 has 50 bases, whereas the template molecule has 75 bases. Since the full sequence of the target molecule is known in advance, it is a simple matter to design the sequence of the template so that the target part and the template will anneal in a desired manner.
  • the template molecule 11 is formed using uracil triphospate (UTP) as a monomer instead of thymine triphosphate (TTP) during the conventional synthesis so that the template will have Us instead of Ts, the Us being positioned in the sequence to align with every A of the complementary part of the target sequence.
  • UTP uracil triphospate
  • TTP thymine triphosphate
  • the template it is normal to construct the template without any Ts at all, but this is not essential. There should, however, be at least one U in the template sequence, and preferably several, e.g. one every two or three nucleotides. Indeed, the template may consist entirely of Us, provided that the target sequence is intended to include a complementary stretch of all As.
  • Step B shows the annealing together of these two molecules.
  • the sequence of the template 11 is made such that it overlaps (i.e. is complementary to) one end of the target part 10, so that both the target part 10 and the template 11 have overhanging non-annealed sections 12 and 13, respectively, and an annealed section 14 of 25 bases each.
  • the resulting annealed construct is then used as a base for polymerization of the remainder of the target molecule 15, extending from the 3' end of the target part 10 in the direction of the arrow X.
  • This can be done by using a DNA polymerization technique, such as that used for PCR employing the enzyme Taq polymerase and a mixture of nucleotide monomers under suitable conditions.
  • the new sequence is complementary to the previously unaligned part 13 of the template molecule 11.
  • Step C the additional bases added in this way produce a target molecule 15 of 100 bases, 50 of which correspond to the sequence of the original target molecule part 10 and the remaining 50 of which are complementary to the overhanging sequence 13 of the template molecule 11.
  • the template molecule 11 is preferably constructed using a dideoxynucleotide at the 3' end of the molecule so that the DNA polymerization reaction does not extend the template itself.
  • the template 1 1 therefore remains shorter than the target molecule 15.
  • the target molecule 15 remains annealed to the template 11 in the form of double-stranded DNA.
  • the template molecule may be digested and disintegrated, for example by the addition of an enzyme (e.g. uracil-N-glycocylase) that cuts the molecule at the U bases.
  • Uracil-N- glycocylase is a commercial enzyme that may be obtained, for example, from Applied Biosystems, USA under trade name AmpErase ® .
  • the reaction is carried out at 50 0 C for 5 minutes and then heated at 95 0 C for 15 to 30 minutes to denature the enzyme.
  • the template is cut into small fragments of the original molecule, the fragments being individual bases or short sequences of two or three bases depending on the number of Us in the template sequence. Since the target molecule 15 does not have any U bases, it remains undigested and exists as single-stranded DNA.
  • the target molecule 15 (which is the molecule with the highest molecular weight in the reaction mixture) may be removed from other components of the digestion product (e.g. digested fragments of the template and unextended target parts) by any suitable method, such as membrane filtration using a 100 base cut-off (e.g. a filter from PSI clone, Princeton Separation, New Jersey, USA), or a gel matrix such as Sephadex ® used for size exclusion. This allows any remaining target parts and small fragments to be separated out, thus providing a purified single-stranded DNA target molecule 15 of 100 bases in length.
  • a 100 base cut-off e.g. a filter from PSI clone, Princeton Separation, New Jersey, USA
  • a gel matrix such as Sephadex ® used for size exclusion
  • the template fragments may re-anneal to complementary parts of the target molecule, these fragments do not adhere strongly (because of the small number of bases in such fragments) and they are normally freed from the target molecule during the separation step.
  • the procedure may be carried out commencing with concentrations or quantities of the starting materials in micro-moles or micro-grams, but increased concentrations or quantities may be employed as required. If it is desired to amplify the resulting target DNA, this may be done by employing conventional polymerase chain reaction, thereby increasing the quantity of the product significantly.
  • the illustrated method may then be repeated using the target molecule of one or two of such procedures as target molecule parts for a further procedure using a suitable new template, thereby creating a target molecule of significantly increased length. Additional repetitions of the procedure will cause the length of the target molecules to increase significantly. Hence, very long target molecules may be formed without incurring unacceptable impurity of the product. This is illustrated in the Table below:
  • mer means a molecule that forms a building block for the polymer structure and is equivalent to the number of bases.
  • sequences up to 1000 bases (1000 mer) are probably most suitable for current practical applications.
  • ESTs expression short tags
  • lengths of one or two kilobases may be desirable.
  • Step A shows the formation of two target molecule parts 10' and 10" (each of 50 bases), and a template 1 1 of 75 bases (again comprising at least one U).
  • the two target molecule parts 10' and 10" may, in particular cases, be identical.
  • the sequence of the template 11 is made such that it will anneal with both of the two target parts 10' and 10" such that the target molecule parts become aligned adjacent to each other as shown in Step B.
  • the two target molecule parts are aligned without a gap, but a gap of one base may be tolerated.
  • the two target molecule parts are then joined by ligation to form the target molecule 15 as shown in Step C.
  • the subsequent steps digestion and purification steps may then be the same as in the embodiment of Fig. 1.
  • the ligation step is carried out in the presence of a suitable ligase enzyme, preferably T4 DNA ligase (which may be obtained, for example, from New England Biolabs Inc. of Ipswich, Massachusetts, USA).
  • T4 DNA ligase which may be obtained, for example, from New England Biolabs Inc. of Ipswich, Massachusetts, USA.
  • This enzyme (which originates from T4 bacteriophage) catalyzes the formation of a phosphodiester bond between juxtaposed 5' phosphate and 3' hydroxyl termini in DNA.
  • LCR ligase chain reaction
  • ATP adenosine triphosphate
  • Most restriction enzyme buffers will be suitable for this purpose.
  • the mixture is incubated at a suitable temperature, optimally 16 0 C, for a suitable time, such as 30 minutes to two hours. Essentially, this reaction forms one piece of DNA from two.
  • the invention allows a relatively short part or parts of a target molecule to be synthesized by conventional techniques, and then enables the full target molecule to be produced in a way that avoids the formation of incorrect or impure sequences. Relatively long sequences can therefore be produced on a reliable basis.
  • Another form of the invention involves a variation of the polymerization process described above. This is illustrated in Fig. 3.
  • the intention is not to produce a single strand of DNA that is necessarily longer than the template molecule, but one that has a different (preferably higher) molecular weight or ionic charge than would normally be the case for a polynucleotide molecule of the same number of bases.
  • an intermediate molecule 100 is provided as well as a template molecule 11.
  • the intermediate molecule and template are of the same length and have complementary sequences.
  • the template 11 is formed with a U in place of at least one T, as in the previous forms of the invention.
  • the intermediate molecule 100 is a strand of conventional DNA produced as single-stranded molecule by conventional DNA synthesis and is normally of less than 100 bases in length, and the template molecule 1 1 is also produced by a conventional method.
  • the intermediate molecule 100 and template 1 1 are allowed to anneal to form a double-stranded molecule, as shown in Step B, which is then provided with a break 101 (or "nick") in the phosphodiester backbone of the molecule by reaction with a deoxyribonuc lease enzyme (e.g. deoxyribonuclease 1, often designated Dnase 1) to produce two fragments 100' and 100", as shown in Step C.
  • a deoxyribonuc lease enzyme e.g. deoxyribonuclease 1, often designated Dnase 1
  • Step D One of the fragments 100' is removed and, as shown in Step D during the polymerization, the remaining fragment 100" is extended by DNA polymerization using deoxynucleotides (dNTPs) of increased molecular weight or ionic charge compared to those forming the intermediate molecule 100 (which are normally "conventional" dNTPs found in nature).
  • dNTPs deoxynucleotides
  • the target 15' is then isolated in the manner indicated above, i.e. by means of digestion of the template and preferably membrane separation/filtration leaving the isolated target molecule 15'.
  • nick translation The process of forming a "nick” in a DNA molecule, followed by DNA polymerization, is sometimes referred to as "nick translation” and is described in Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Horbor laboratory, Cold Spring Harbor, N. Y., 2001 (the disclosure of which is incorporated herein by reference), and further information can be obtained from Kelly, W.S., Chalmers, K and Murray, N.E., Isolation and characterization of a lambdapolA transducing phage; Proc. Natl. Acad. Sci., USA, Vol. 74, No. 12, pp 5632-5636 (December 1977) (the disclosure of which is also incorporated herein by reference).
  • the intermediate target strand of the annealed construct is nicked or cut by the Dnase 1.
  • DNA polymerase e.g. E. coli polymerase 1
  • DNA polymerase removes intermediate molecule part 100'
  • DNA polymerase adds deoxynucleotides to the DNA molecule part 100" at the 3 '-end of the cut or nick.
  • the net effect of this cutting, removal and addition of deoxyribonucleotides is a generalized replacement of part 100' by extension of part 100" thus forming molecule part 102.
  • these monomers become incorporated into the molecule part 102 being added to the fragment 100" of original intermediate target molecule.
  • Dnases deoxyribonucleases
  • substrate specificities and may be sequence-specific, so that the break may be produced at a precise location within the intermediate target molecule 100; however, this is not essential.
  • a Dnase that is specific for single-stranded molecules is employed in this form of the invention.
  • deoxyribonuc lease 1 cleaves DNA preferentially at phosphodiester linkages adjacent to a pyrimidine nucleotide, yielding 5'-phosphate terminated polynucleotides with a free hydroxyl group on position 3', on average producing tetranucleotides. It acts on single-stranded DNA.
  • modified nucleotides that can be incorporated into the target part 102.
  • the modifications to the nucleotides occur within either the nucleotide base, the ribose, or the phosphate to increase molecular weight or ionic charge. Examples include adenosine-5'-(l-thiotriphosphate) and cytidine-5'-(l-thiotriphoshpate).
  • Modified nucleotides with 5-fluorescein (MW 816.7) 6-carboxyfluorescein (MW 537.5) and Texas red (MW 744.1) can be purchased from number of vendors (e.g. Integrated DNA Technologies, IL, USA).
  • dNTPs of increased ionic charge may be incorporated into the target molecule in the same way.
  • Molecules of different ionic charge act differently during gel separation by means of electrophoresis, and can thus improve methods of separation.
  • Fig. 4 illustrates a variation of the procedure of Fig. 3 which produces a target molecule 15" that is both long (e.g. having more than 100 bases) and of increased molecular weight or ionic charge. With increased length, there is a greater risk of a cut or nick being formed at more than one location in the molecule.
  • Step A an intermediate molecule 100" is provided that is longer than the template molecule 11 , but that has a part that is complementary to template 11.
  • Step B these molecules are allowed to anneal. Being longer, part of the intermediate molecule 100" overhangs the template 11 at the 3' end of the template, as shown (the respective sequences are chosen to produce such a mutual alignment).
  • Step C involves contacting the annealed construct with Dnase 1 used at very low concentration (e.g. 2 ⁇ l of DNase 1 solution with DNase 1 at 0.5 U/ ⁇ l) so that there is minimal cutting of the intermediate molecule, i.e. preferably one or two nicks per intermediate molecule. Normally, there is just one nick formed for every 300-500 bases at low concentration. There may additionally be nicking of the template molecule 11.
  • Dnase 1 used at very low concentration (e.g. 2 ⁇ l of DNase 1 solution with DNase 1 at 0.5 U/ ⁇ l) so that there is minimal cutting of the intermediate molecule, i.e. preferably one or two nicks per intermediate molecule. Normally, there is just one nick formed for every 300-500 bases at low concentration. There may additionally be nicking of the template molecule 11.
  • Fig. 4 shows two versions of the nicked construct labeled X and Y, i.e. one (X) having two nicks 105 and 106 in the intermediate molecule 100" forming fragments 120 and 121 with about 30 nucleotides corresponding to the annealing region of the template molecule 11 and a longer fragment 122, together with one nick 107 in the template 1 1, and another construct (Y) having just one nick 105 in the intermediate molecule 100" forming fragments 122 and 123, and no nicks in the template molecule 1 1.
  • Step D Polymerization is then commenced as shown in Step D.
  • a high temperature e.g. a melting temperature Tm of 8O 0 C
  • nick fragments with short annealing segments e.g. 120 and 121
  • Tm melting temperature
  • polymerize thus leaving a desired nick fragment 122 bound to the template and polymerized, thus obtaining the extended target 15" containing a section 102' containing nucleotides of higher molecular weight or ionic charge than those in the intermediate molecule 100".
  • the replacement of part 123 with part 102' is the same as for the embodiment of Fig. 3. In both cases, therefore, a target molecule 15" is formed, so the additional nicking does not affect the result.
  • the template may be digested as before and the products subjected to filtration to isolate the single-stranded target molecule 15", as shown in Step E.
  • a 50 mer oligonucleotide is synthesized using solid phase synthesis in a nucleic acid synthesizer (Integrated DNA Technologies, Illinois, USA).
  • the template sequence is:
  • a 50 mer oligonucleotide is synthesized using solid phase synthesis using solid phase synthesis in a nucleic acid synthesizer (Integrated DNA Technologies, Illinois, USA).
  • the target sequence is:
  • a. Reactions are carried out in two 1.5 ml Eppendorf tubes as follows: i. Template oligonucleotide (20 ⁇ M) - 100 ⁇ l ii. Target oligonucleotide (20 ⁇ M) - 100 ⁇ l iii. Buffer (10X) - 50 ⁇ l iv. dNTP (3.12 nM) - 20 ⁇ l v. DNA polymerase - 10 units vi. Water (to 500 ⁇ l). b. Incubated at 37 0 C for 1 hour.
  • Reagents iii, iv, v are from Applied Bisosystems, USA, and reagent vi is from Invitrogen, USA.
  • f Add 250 ⁇ l volume of PCR binding buffer to 250 ⁇ l volume of reaction product, and mix by pipetting 3 times. g. Place a single filter column in a 2ml wash tube and transfer the DNA mixture to the column (filter) and centrifuge at 6000rpm for 1 min. h. Add 400 ⁇ l wash buffer to the filter column and centrifuge at 6000rpm for lmin. Discard the filtrate, i. Repeat step (j) once more. j. Centrifuge the column at 14000 rpm for 2min to ensure filter is dry. k. Add 50 ⁇ l elution buffer and leave for 5min.
  • a 40 mer oligonucleotide AND A 50 mer oligonucleotide is synthesized using solid phase synthesis in a nucleic acid synthesizer (Integrated DNA Technologies, Illinois, USA).
  • the template sequence is:
  • a 50 mer oligonucleotide is synthesized using solid phase synthesis using solid phase synthesis in a nucleic acid synthesizer (Integrated DNA Technologies, Illinois, USA).
  • the target sequence is:

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Abstract

Cette invention concerne un procédé de production d’ADN monobrin. Dans une forme de l’invention, le procédé consiste à fournir une molécule matricielle oligonucléotidique contenant de l’uracile ayant une séquence complémentaire d’une partie d’une molécule d’ADN monobrin cible d’une longueur supérieure à la molécule matricielle; à fournir une ou plusieurs parties de la molécule cible comprenant une séquence de base complémentaire d’une partie de la molécule matricielle; à hybrider la/les partie(s) de la molécule cible à la molécule matricielle et à former la molécule cible entière en ligaturant au moins deux parties adjacentes de la molécule cible alors hybridée à la molécule matricielle, et/ou à allonger au moins une partie de la molécule cible pour former une séquence complémentaire du reste de la molécule matricielle par polymérisation nucléotidique, puis à séparer la molécule matricielle de la molécule cible. Dans une autre forme de l’invention, une molécule intermédiaire est hybridée à la matrice avant d'être coupée par voie enzymatique, et une partie est ensuite allongée par polymérisation de l’ADN à l'aide des monomères de poids moléculaire ou de charge ionique accrus par rapport aux monomères utilisés pour former la molécule intermédiaire.
PCT/CA2008/000224 2008-02-07 2008-02-07 Synthèse d’adn monobrin Ceased WO2009097673A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018011067A3 (fr) * 2016-07-11 2018-02-22 Glaxosmithkline Intellectual Property Development Limited Nouveaux procédés de production d'oligonucléotides

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WO2001064864A2 (fr) * 2000-02-28 2001-09-07 Maxygen, Inc. Recombinaison et isolement de fragments d'acides nucleiques a l'aide de matrices d'acides nucleiques simple brin
US6355484B1 (en) * 1994-02-17 2002-03-12 Maxygen, Inc. Methods and compositions for polypeptides engineering
WO2004083427A2 (fr) * 2003-03-20 2004-09-30 Nuevolution A/S Codage par ligature de petites molecules
US20080118915A1 (en) * 2006-08-14 2008-05-22 Thuraiayah Vinayagamoorthy Synthesis of single-stranded dna

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6355484B1 (en) * 1994-02-17 2002-03-12 Maxygen, Inc. Methods and compositions for polypeptides engineering
WO2001064864A2 (fr) * 2000-02-28 2001-09-07 Maxygen, Inc. Recombinaison et isolement de fragments d'acides nucleiques a l'aide de matrices d'acides nucleiques simple brin
WO2004083427A2 (fr) * 2003-03-20 2004-09-30 Nuevolution A/S Codage par ligature de petites molecules
US20080118915A1 (en) * 2006-08-14 2008-05-22 Thuraiayah Vinayagamoorthy Synthesis of single-stranded dna

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018011067A3 (fr) * 2016-07-11 2018-02-22 Glaxosmithkline Intellectual Property Development Limited Nouveaux procédés de production d'oligonucléotides
GB2558335A (en) * 2016-07-11 2018-07-11 Glaxosmithkline Intellectual Property Ltd Novel processes for the production of oligonucleotides
GB2558335B (en) * 2016-07-11 2019-12-04 Glaxosmithkline Intellectual Property Ltd Novel processes for the production of oligonucleotides
RU2719641C1 (ru) * 2016-07-11 2020-04-21 Глаксосмитклайн Интеллекчуал Проперти Дивелопмент Лимитед Новые способы получения олигонуклеотидов
US10640812B2 (en) 2016-07-11 2020-05-05 Glaxosmithkline Intellectual Property Development Limited Processes for the production of oligonucleotides
EP3481838B1 (fr) 2016-07-11 2020-08-19 GlaxoSmithKline Intellectual Property Development Limited Nouveaux procédés de production d'oligonucléotides
EP3702360A1 (fr) * 2016-07-11 2020-09-02 GlaxoSmithKline Intellectual Property Development Limited Nouveaux procédés de production d'oligonucléotides
CN115161366A (zh) * 2016-07-11 2022-10-11 葛兰素史克知识产权开发有限公司 生产寡核苷酸的新颖方法
US12275983B2 (en) 2016-07-11 2025-04-15 Glaxosmithkline Intellectual Property Development Limited Processes for the production of oligonucleotides
CN115161366B (zh) * 2016-07-11 2025-08-05 葛兰素史克知识产权开发有限公司 生产寡核苷酸的新颖方法

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