Compositions and methods for enhanced synthesis of nucleic acid molecules

• US 20040265969A1
• Filed: 07/27/2004
• Published: 12/30/2004
• Est. Priority Date: 03/13/1998
• Status: Active Grant

• Current Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Gebeyehu, Gulilat, Jessee, Joel A., Schuster, David, Li, Wu-Bo, Xia, Jiulin
• Granted Patent: US 7,344,863 B2
• Time in Patent Office: Days
• US Class Current: 435/69.1
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer

Abstract

The present invention is directed to compositions and methods for enhancing synthesis of nucleic acid molecules, particularly GC-rich nucleic acid molecules. Specifically, the invention provides compositions comprising one or more nitrogen-containing organic compounds having a formula selected from the group consisting of formula I and formula II (or salts or derivatives thereof), preferably 4-methylmorpholine N-oxide or betaine (carboxymethyltrimethylammonium), and further comprising one or more compounds selected from the group consisting of proline and an N-alkylimidazole compound, and more preferably proline, 1-methylimidazole or 4-methylimidazole. The invention further relates to methods for enhanced, high-fidelity synthesis of nucleic acid molecules, including via amplification (particularly PCR), reverse transcription, and sequencing methods. The invention also relates to nucleic acid molecules synthesized by these methods, to fragments or derivatives thereof, and to vectors and host cells comprising such nucleic acid molecules, fragments, or derivatives. The invention also relates to kits for synthesizing, amplifying, reverse transcribing or sequencing nucleic acid molecules comprising one or more of the compositions of the invention.

Claims

1. A method for producing a polypeptide, comprising:

(a) mixing a nucleic acid template with one or more compounds having a chemical formula selected from the group consisting of formula I or formula II, or a salt thereof, wherein said compound is not betaine;

where A is

where X is

where N is positively charged;

wherein q=1 to 100,000, wherein when q=2 to 100,000 each monomer of formula I may be the same as or different from the other monomers of formula I;

wherein Z may be the same as or different from Y;

wherein each Y and Z are independently selected from the group consisting of —

OH, —

NH₂, —

SH, —

PO₃H, —

CO₂H, —

SO₃H and hydrogen;

wherein f is an integer from 0 to 2, m is an integer from 0 to 20 and e is an integer from 0 to 2;

wherein R₄, R₅, and R₆ may be the same or different and are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, amino, mercaptan, thiol, halo, nitro, nitrilo, hydroxy, hydroxyalkyl, hydroxyaryl, phosphato, alkoxy, oxide, ether, ester (alkanoyloxy), carboxy, carbonyl, sulfonyl, sulfonic and amido groups, and d is an integer from 0 to 2;

wherein a, b, and c are independently an integer from 0 to 1, with the proviso that no more than two of a, b, and c are zero;

wherein R₁, R₂ and R₃ may be the same or different and are independently selected from the group consisting of;

with the proviso that no more than two of A, R₁, R₂ and R₃ are selected from the group consisting of hydrogen, methyl, ethyl and propyl; and

with the proviso that if one, and only one, of R₁, R₂ and R₃ is ═

O, then A is none of hydrogen, methyl, ethyl and propyl;

wherein each R₇ and W may be the same or different and are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, amino, thiol, mercaptan, halo, nitro, nitrilo, hydroxy, hydroxyalkyl, hydroxyaryl, phosphato, alkoxy, oxide, ether, ester (alkanoyloxy), carboxy, carbonyl, sulfonyl, sulfonic and amido groups;

g is an integer from 0 to 2 and n is an integer from 0 to 20, with the proviso that if two of R₁, R₂, and R₃ are ═

O, then the other is not ═

O;

wherein Formula II is saturated or unsaturated;

wherein q=1 to 100,000, wherein when q=2 to 100,000, each monomer of formula II may be the same as or different from each other monomer of formula II;

wherein X is selected from the group consisting of N, C, O, P and S;

wherein Y is selected from the group consisting of O, N, S, P, C, —

O—

NH—

, —

O—

CH₂—

NH—

, —

O—

CH₂—

O—

, —

NH—

CH₂—

NH—

, —

O—

CH(CH₃)—

NH—

, —

NH—

CH(CH₃)—

NH—

, —

O—

CH(CH₃)—

O—

, —

NH—

C(CH₃)₂—

NH—

, —

O—

S—

, —

O—

CH₂—

S—

, —

NH—

S—

, —

NH—

CH₂—

S—

, and other mercaptan, phosphato, alkoxy, oxide, ether, esters (alkanoyloxy), carboxy, sulfonyl, sulfonic and amido groups;

with the proviso that if either X or Y is N, then the other is not C;

wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇ and R₈ may be the same or different and are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, amino, thiol, mercaptan, halo, nitro, nitrilo, hydroxy, hydroxyalkyl, hydroxyaryl, phosphato, alkoxy, oxide, ether, ester (alkanoyloxy), carboxy, sulfonyl, sulfonic and amido groups; and

wherein a, b, c, d, e, m, n and o are integers which may be the same or different and are independently selected from 0 to 2 for a, b, c, d, and e, and 0 to 5 for m, n and o, to form a mixture;

(b) incubating the mixture under conditions whereby a first nucleic acid molecule complementary to all or a portion of the template and encoding at least one polypeptide is made;

(c) inserting one or more of the first nucleic acid molecules into a host cell; and

(d) expressing the polypeptide encoded by the first nucleic acid molecule in the host cell.

2. The method of claim 1, further comprising isolating the polypeptide from the host cell.

3. The method of claim 1, wherein the bases of the nucleic acid template comprise greater than about 60% guanine and cytosine.

4. The method of claim 1, wherein said insertion occurs via calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction or infection.

5. The method of claim 1, further comprising cloning the first nucleic acid molecule into a vector or plasmid prior to insertion into the host cell.

6. The method of claim 2, wherein said isolation occurs via ammonium sulfate precipitation, ethanol precipitation, electrophoresis, immunoadsorption or chromatography.

7. The method of claim 1, wherein the host cell is an Escherichia coli host cell.

8. A polypeptide produced by the method of claim 1.

9. A method for producing a polypeptide, comprising:

(a) mixing a nucleic acid template with one or more compounds having a chemical formula selected from the group consisting of 1-methylimidazole, 4-methylimidazole, 4-methylmorpholine N-oxide, poly(2-ethyl-2-oxazoline) of average molecular weight about 50,000 to about 500,000 daltons and poly(diallyldimethylammonium chloride) of average molecular weight about 100,000 to about 200,000 daltons;

(b) incubating the mixture under conditions whereby a first nucleic acid molecule complementary to all or a portion of the template and encoding at least one polypeptide is made;

(c) inserting one or more of the first nucleic acid molecules into a host cell; and

(d) expressing the polypeptide encoded by the first nucleic acid molecule in the host cell.

10. The method of claim 9, further comprising isolating the polypeptide from the host cell.

11. The method of claim 9, wherein the bases of the nucleic acid template comprise greater than about 60% guanine and cytosine.

12. The method of claim 9, wherein said insertion occurs via calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction or infection.

13. The method of claim 9, further comprising cloning the first nucleic acid molecule into a vector or plasmid prior to insertion into the host cell

14. The method of claim 10, wherein said isolation occurs via ammonium sulfate precipitation, ethanol precipitation, electrophoresis, immunoadsorption or chromatography.

15. The method of claim 9, wherein the host cell is an Escherichia coli host cell.

16. A polypeptide produced by the method of claim 9.