Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-06-24
2003-08-12
Siew, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C435S091500, C435S183000, C435S193000, C536S022100, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330, C530S350000
Reexamination Certificate
active
06605428
ABSTRACT:
The present invention relates to a method for the direct, exponential amplification and sequencing of DNA molecules as well as the use of the method. The direct, exponential amplification and sequencing of DNA molecules is referred to as “DEXAS” in the following.
DESCRIPTION OF THE RELATED ART
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.
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.
Many DNA polymerases, including the Taq DNA polymerase, that are 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, ddCTP, ddGTP, 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 the methods described above require an interruption between the first step of exponential amplification of the template DNA and the second step for the synthesis of truncated DNA molecules and also 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.
SUMMARY OF THE INVENTION
The object of the present invention is to provide an improved, rapid and reliable method for sequencing DNA molecules, preferably genomic DNA.
A further object of the present invention is to provide a direct method for nucleic acid sequencing which simultaneously increases the exponential amplification of molecules of full length as well as of molecules of truncated length in the reaction 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 an improved, rapid and reliable method for sequencing DNA molecules, preferably genomic DNA that can be carried out in a single step in a single container.
A further object of the present invention is to provide an application according to the invention for sequence determination in medical diagnostics, forensics and population genetics.
Further objects of the invention are obvious to a person skilled in the art from the description.
In contrast to the above-described “CAS” method a DNA polymerase is used as the thermostable DNA polymerase which, compared to wild-type Taq DNA polymerase, has a reduced discrimination against the four ddNTPs in the buffer and under the conditions that are used for the thermocycling. More preferably a DNA polymerase is used which carries a “Tabor-Richardson” mutation or a functional derivative thereof which also has no 5′-3′exonuclease activity such as e.g. AmplitaqFS( (Taq DNA polymerase (-exo5′-3′)(F667Y), Tabor and Richardson (1995), loc. cit.), Taquenase( (Taq DNA polymerase (235 (-exo5′-3′) (F667Y), Tabor and Richardson (1995), loc. cit.) and Thermo Sequenase( (Taq DNA polymerase (-exo5′-3′) (F667Y), Tabor and Richardson (1995), loc. cit.) as well as mixtures thereof or other DNA polymerases and mixtures thereof which are thermostable can also be used in the method of the present invention. Surprisingly the use of a DNA polymerase which, in comparison to wild-type Taq DNA polymerase, has a reduced discrimination against the four ddNTPs, enables the simultaneous and exponential synthesis of truncated as well as of full fragments from the start of the cycling reaction. Hence the present invention concerns a method for the direct sequencing of a nucleic acid molecule from a complex mixture of nucleic acids, such as e.g. total genomic human DNA, containing a reaction buffer, deoxynucleotides or derivatives thereof and a dideoxynucleotide or another terminating nucleotide and a thermostable polymerase which has a reduced discrimination against ddNTPs in comparison to wild-type Taq DNA polymerase. Within the sense of the present invention direct sequencing means that the nucleic acid fragment to be sequenced is simultaneously amplified and sequenced in one step without interrupting the reaction and without prior amplification of the nucleic acid fragment to be sequenced by the known methods and in such a manner that an unequivocal sequence ladder is readable.
A further difference between DEXAS and the “CAS” method described above is the principle that the initial and subsequent cycle sequencing reaction is carried out with two primers, a first primer, and a second primer which lies on the strand complementary to the first, which are preferably present in a non-equimolar ratio and serve to simultaneously produce adequate template molecules of full length as well as truncated molecules which contribute to the sequencing of the DNA molecule. Four reactions are prepared, one for the determination of each base, so that each reaction contains two primers preferably in a non-equimolar ratio to one another of which either one is labelled and the other is unlabelled or both are differently labelled. The said non-equimolar ratio between the first primer and the second primer enables the simultaneous and exponential synthesis of the truncated as well as of the full fragments from the start of the cycling reaction. Furthermore each reaction contains from the start the DNA template to be sequenced as well as a buffer solution, thermostable DNA polymerase, thermostable pyrophosphatase (
Kilger Christian
Paabo Svante
Arent Fox Kintner & Plotkin & Kahn, PLLC
Roche Diagnostics GmbH
Siew Jeffrey
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