Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-02-02
2001-08-07
Campbell, Eggerton A. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200
Reexamination Certificate
active
06270962
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates primarily to improvements in methods of DNA sequencing. In particular, the invention relates primarily to the elimination of stops or pauses in chain termination methods of DNA sequencing by the addition of nitrogen-containing organic compounds such as betaine, trimethylamine N-oxide and dimethylglycine. The invention also provides for DNA sequencing kits containing these compounds. The invention also provides for improvements in other laboratory procedures using DNA polymerases, such as polymerase chain reaction (PCR).
Efficient DNA sequencing technology is very important to the development of the biotechnology industry as well as for basic biological research. Improvements in both efficiency and accuracy of DNA sequencing are needed to keep pace with the demands for DNA sequence information. The Human Genome Project, for example, has set a goal for dramatically increasing the efficiency, cost-effectiveness and throughput of DNA sequencing techniques. (See Collins, F., and Galas, D. (1993)
Science
262:43-46.)
Most DNA sequencing today is carried out by a chain termination method of DNA sequencing. The most popular chain termination methods are variants of the dideoxynucleotide-mediated chain termination method of Sanger (see Sanger et al. (1977)
Proc. Nat. Acad. Sci., USA
74:5463-5467). Thousands of laboratories employ this technique including those doing automated sequencing for the Human Genome Project. Commercial kits containing the reagents needed for this method of DNA sequencing are available and are widely used.
Although commonly used, the Sanger (dideoxy) sequencing technique has problems and limitations. One of the major problems with this method is the incidence of DNA polymerase stops or pauses which interfere with the determination of the DNA sequence. Stops are predominantly problematic in regions of the DNA that are GC-rich or in regions that are especially far from the primer. In addition, stops occur more frequently in impure DNA preparations. Because of this, DNA purification is generally required before DNA can be sequenced by the dideoxy method.
Various methods have been proposed to eliminate stops in dideoxy sequencing. For example, researchers have tried varying the reaction temperature, using a variety of DNA polymerases, stabilizing the DNA polymerase, and extending the prematurely terminated DNA molecules with terminal deoxynucleotidyl transferase (see T. W. Fawcette and S. G. Bartlett (1990)
BioTechniques
9:46-48; D. Pisa-Williamson and C. W. Fuller (1992) United States Biochemical Corp. Comments 19:29-36; J. Sambrook, E. F. Fritsch and T. Maniatis, ed. (1989)
Molecular Cloning: A Laboratory Manual
, second edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.; and F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith and K. Struhl, ed. (1989)
Current Protocols in Molecular Biology
, Greene Publishing Associates and Wiley-Interscientific, John Wiley and Sons, N.Y.) However, none of these methods has been reliable. There is a continuing need to eliminate the problem of stops in DNA sequencing and thereby improve the efficiency and cost-effectiveness of this important process.
SUMMARY OF THE INVENTION
This invention provides for methods of decreasing the incidence of DNA polymerase stops that occur in a reaction mixture containing a DNA polymerase. The incidence of DNA polymerase stops is decreased by the addition of nitrogen-containing organic molecules, which are described herein, to the reaction mixture. These compositions of the invention are added to the reaction mixture in an amount that is effective to decrease the incidence of DNA polymerase stops. The reaction mixture can be used in a chain termination method of DNA sequencing. Preferably the chain termination method of DNA sequencing is a dideoxynucleotide DNA sequencing method. The reaction mixture can also be a PCR reaction mixture. The DNA which is sequenced or amplified can be purified or unpurified. Unpurified DNA can be present, for example, as a crude cell lysate.
The method of decreasing the incidence of DNA polymerase stops can be combining, in an aqueous solution, a DNA molecule, a DNA polymerase or DNA polymerases, a mixture of deoxyribonucleoside triphosphates, a chain elongation inhibitor, and one or more of the compositions of the invention to form a reaction mixture. The DNA polymerase is capable of producing a nucleic acid complementary to a portion of the DNA molecule by using the DNA molecule as a template. The reaction mixture is incubated to allow the DNA polymerase to form nucleic acid fragments of varying length by using the DNA molecule as a template. The nucleic acid fragments that are formed are complementary to the DNA molecule that is being sequenced.
In some variations of the above method, a nucleic acid primer is also added to the reaction mixture. This primer is complementary to a second portion of one strand of the DNA molecule that is located downstream from the first portion of that strand. The DNA polymerase that is used is capable of extending the 3′ end of the primer to produce a nucleic acid that is complementary to the first portion of the DNA molecule.
The invention provides improved methods for sequencing a DNA molecule by the chain termination method, wherein the DNA molecule is combined with a DNA polymerase capable of producing a nucleic acid complementary to a portion of the DNA molecule by using the DNA molecule as a template; with a mixture of deoxyribonucleoside triphosphates; and with chain elongation inhibitor, to form a reaction mixture. This reaction mixture is incubated to permit the DNA polymerase to form nucleic acid fragments of varying length by using the DNA molecule as a template, wherein the nucleic acid fragments are complementary to the DNA molecule. The improved part of this method is the addition to the reaction mixture of an amount of one or more of the nitrogen-containing organic compounds described herein.
The invention also provides for kits for DNA sequencing by the chain termination method. These kits have instructional material, a container which contains one or more deoxyribonucleoside triphosphates, a container which contains a chain elongation inhibitor, and an amount of the compositions of the invention. The kits may also contain other components. Preferably, the kits are for DNA sequencing by the dideoxy DNA sequencing method.
REFERENCES:
patent: 5545539 (1996-08-01), Miller
patent: 44 11 588 C1 (1995-09-01), None
patent: WO 96/12041 (1996-04-01), None
Baskaran, Namadev, et al.,Genome Research,6:633-638 (1996).
Del Sal, Giannino, et al.,BioTechniques,7(5):514-519 (1989).
Melchior, William B., et al.,Proc. Nat. Acad. Sci. USA,70(2):298-302 (1973).
Pisa-Williamson, Denise, et al., U.S. Biochemical Corp. (Editorial Comments), 19(2):29-36 (1992).
Rees, William A., et al.,Biochemistry,32:137-144 (1993).
Sanger, F., et al.,Proc. Natl. Acad. Sci. USA,74:5463-5467 (1977).
Santoro, Marcelo M., et al.,Biochemistry,31:5278-5283 (1992).
Winship, P.R., “An improved method for directly sequencing PCR amplified material using dimethyl sulphoxide,”Nucleic Acids Research,17(3):1266 (1989).
Thakar, M. et al., “Osmolyte Mediation of T7 DNA Polymerase and Plasmid DNA Stability,”Biochemistry,33:12255-12259 (1994).
Chamberlin Michael
Mytelka Daniel
Campbell Eggerton A.
Pletta Gregory T.
Quarles & Brady LLP
The Regents of the University of California
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