Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1997-12-16
2001-05-01
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S091100, C435S810000
Reexamination Certificate
active
06225092
ABSTRACT:
The present invention relates to a method for the uncoupled, direct, exponential amplification and sequencing of DNA molecules by the addition of a second thermostable DNA polymerase and it also relates to the application of the said method. The uncoupled, direct, exponential amplification and sequencing of DNA molecules by the addition of a second thermostable DNA polymerase is referred to as “DEXTAQ” in the following.
TECHNICAL FUNDAMENTALS
The DNA sequence determination as developed by Sanger et al. ((1977)
Proc. Natl. Acad Sci. USA
74, 5463-5467) is usually carried out with a T7 DNA polymerase (Tabor S. and Richardson, C. C. (1989)
Proc. Natl. Acad Sci. USA
86, 4076-4080). This method requires relatively large amounts of a purified, single-stranded DNA template. Recently cycle sequencing has been developed (Murray, V. (1989)
Nucleic Acids Res
. 17, 8889). This method does not require a single-stranded template and allows the sequence reaction to be initiated with relatively small amounts of template. However, the template DNA has to be purified to almost complete homogeneity and is usually prepared by means of cloning in plasmids (Bolivar, F. et al., (1977)
Gene
2, 95-113) and subsequent plasmid purification (Birnboim, H. C. and Doly, J. (1979)
Nucleic Acids Res
. 7, 1513-1523) or by means of PCR amplification (Mullis, K. B. and Faloona, F. A. (1987)
Methods Enzymol
. 155, 335-350). Only one primer is used in both of the methods described above.
Known thermostable polymerases that are used for cycle sequencing e.g. ThermoSequenase and Taquenase carry a mutation which is known as the “Tabor Richardson” mutation (Tabor, S. & Richardson, C. C. (1995)
Proc. Natl. Acad. Sci. USA
92, 6339-6343) in which a tyrosine is present instead of a phenylalanine in the crevice of the enzyme which, during polymerization of the DNA molecule being formed, is responsible for discriminating between the incorporation of either deoxynucleotides or dideoxynucleotides. Such enzymes or functional derivatives thereof have an increased ability to incorporate dideoxynucleotides into DNA fragments that are being formed and can be used to improve the signal uniformity in sequencing reactions. The increased ability of the aforementioned DNA polymerases with a Tabor-Richardson mutation to incorporate dideoxy-nucleotides increases the statistical probability that a chain termination occurs due to incorporation of a dideoxynucleotide into a DNA molecule being formed.
In one embodiment of the cycle sequencing which is referred to as “coupled amplification and sequencing” or “CAS” Ruano and Kidd ((1991)
Proc. Natl. Acad Sci. USA
88, 2815-2819; U.S. Pat. No. 5,427,911) have shown that one can use a two-step protocol to generate sequences from DNA templates. In the first step 15 PCR cycles are carried out with Taq DNA polymerase in the absence of dideoxynucleotides in order to prepare an adequate amount of sequencing template. In a second step in which dideoxynucleotides and a labelled primer are added, CAS produces the sequence as well as the additional amplification of the target sequence. Two primers are used in both steps of the method.
Taq DNA polymerase, that is used in coupled DNA sequencing reactions strongly discriminates against ddNTPs and preferably incorporates dNTPs if it is furnished with a mixture of ddNTPs as well as dNTPs. In addition, it incorporates each ddNTP, i.e. ddATP, ddGTP, ddCTP, ddTTP, with a strongly varying efficiency. Hence the optimization of the CAS process requires careful titration of the dideoxynucleotides.
Furthermore since coupled amplification and sequencing depends on the amount of the initial DNA, the distance between the two primers and the concentrations and the ratios of the ddNTPs and dNTPs relative to one another and to each other, the optimization of coupled amplification and sequencing reactions (CAS) requires that the reaction conditions are individually optimized for a particular DNA fragment.
All methods described above require a separate step for template production, CAS accomplishes this with an interruption between the first step for the exponential amplification of the template DNA and the second step for the synthesis of truncated DNA molecules. Also, all methods require the individual optimization of a given DNA fragment which can be tedious and time-consuming and can lead to errors especially when sequencing a large number of different DNA molecules or when processing large amounts of samples in a hospital or laboratory or when sequencing rare samples for forensic or archaeological studies.
For this reason it would be advantageous to have available a method for sequencing nucleic acids which simultaneously potentiates the exponential amplification of molecules of full length and of molecules of truncated length in the reaction which leads to a reduction of the required amount of starting nucleic acid molecules and does not require an interruption of the exponential amplification step and of the sequencing step so that the whole reaction can be carried out more rapidly and with fewer manipulations.
Furthermore it would also be advantageous to have available a method for sequencing nucleic acid molecules which allows an increase in the distance between the positions of the two primers on the nucleic acid molecule to be sequenced, is relatively independent of the distance between the said primers and in general does not require an optimization of the reaction conditions for each DNA fragment to be sequenced.
SUMMARY OF THE INVENTION
An object of the present invention is to provide an improved, rapid and reliable method for sequencing nucleic acid molecules.
A further object of the present invention is to provide a method for sequencing nucleic acid molecules that can be carried out in an uninterrupted manner, in a single step and in a single container.
A further object of the present invention is to provide a nucleic acid sequencing which simultaneously increases the exponential amplification of molecules of full length as well as of molecules of truncated length which leads to a reduction of the initial amount of nucleic acid molecules that are required for the cycling reaction.
A further object of the present invention is to provide a method for sequencing nucleic acid molecules which leads to an increase in the distance at which both primers can be positioned on the nucleic acid molecule to be sequenced.
A further object of the present invention is to provide a method for sequencing a nucleic acid which increases the signal-to-noise ratio of specific, correctly terminated molecules to unspecifically terminated molecules.
A further object of the present invention is to provide an application of the method according to the invention for sequence determination in medical diagnostics, forensics and population genetics.
Further objects of the invention can be deduced by a person skilled in the art from the description.
The thermocycling reaction of the present invention comprises a first primer and a second primer which serve to simultaneously produce sufficient template molecules of full length as well as molecules of truncated length which contribute to the sequencing of the nucleic acid molecule. Either one primer is labelled and the other is not or both are differently labelled. In addition each reaction initially contains the nucleic acid template to be sequenced as well as a buffer solution and the four deoxynucleotides or derivatives thereof and one dideoxynucleotide or another terminating nucleotide e.g. 3′-aminonucleotides or 3′-ester-derivatized nucleotides. A thermostable pyrophosphatase can be optionally added. Four reaction mixtures are prepared one for the determination of each base.
However, in contrast to the methods known in the state of the art, it was surprisingly found that direct, exponential amplification and sequencing can be carried out by adding two different types of DNA polymerases to the initial cycle sequencing reaction: a first thermostable DNA polymerase and a second thermostable DNA polymerase with a red
Kilger Christian
P{umlaut over (aa)}bo Svante
Arent Fox Kintner & Plotkin & Kahn, PLLC
Horlick Kenneth R.
Roche Diagnostics GmbH
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