Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-05-24
2002-06-04
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091500, C435S091200
Reexamination Certificate
active
06399304
ABSTRACT:
The present invention relates to a method involving sequential activation of one or more enzymatic activities within a reaction as e.g. sequencing, amplification or uncoupled, direct, exponential amplification and sequencing of DNA molecules, coupled reversed transcription and amplification reactions wherein all these reactions contain at least two different enzyme activities.
Especially, the present invention relates to a method for the uncoupled, direct, exponential amplification and sequencing of DNA molecules in a thermocycling reaction which initially comprises a nucleic acid molecule, a first primer, a second primer, a reaction buffer, deoxynucleotides or derivatives thereof and at least one dideoxynucleotide or another terminating nucleotide wherein the thermocycling reaction contains at least two thermostable DNA polymerases with different properties for incorporating dideoxynucleotides wherein one of the two thermostable DNA polymerases is activated in a later cycle of the thermocycling reaction than the other thermostable DNA polymerase. The present invention further relates to the use of a second primer pair called “silent primers” which comprises a third and eventually a forth primer, which are not labeled or differently labeled, compared to the first primer pair at least one of which is labeled or biotinylated.
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.
In one embodiment of 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 discriminate against ddNTPs and preferably incorporate dNTPs if they are furnished with a mixture of ddNTPs as well as dNTPs. 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, the optimization of coupled amplification and sequencing reactions (CAS) requires that the reaction conditions are individually optimized for a particular DNA fragment.
Other known thermostable polymerases that are used for 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 cleft 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.
Therefore it would be expected that the use of a thermostable polymerase which carries a Tabor-Richardson mutation would limit the distance at which the two primers could be placed on the DNA molecule to be sequenced. This in turn restricts the choice of primers that can be used in a given sequencing reaction.
All the methods described above require 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 and they 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 a method for sequencing nucleic acids was developed which simultaneously potentiates the exponential amplification of molecules of complete length and of molecules of shortened 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 (EP 0 849 364 and EP 0 854 196).
Methods for sequencing nucleic acid molecules were developed (called DEXAS and DEXTAQ, respectively), which allows an increase in the distance between the positions of the two primers on the nucleic acid molecule to be sequenced (EP 0 849 364 and EP 0 854 196). These methods are 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.
DEXAS and DEXTAQ are rapid and reliable methods for sequencing nucleic acid molecules that can be carried out in an uninterrupted manner, in a single step and in a single container. DEXAS and DEXTAQ simultaneously increase the exponential amplification of molecules of complete length as well as of molecules of shortened length which leads to a reduction of the initial amount of nucleic acid molecules that are required for the cycling reaction.
In contrast to the DEXAS method that makes use of one enzyme having reduced discrimination against ddNTPs compared to wild-type Taq DNA polymerase in the buffer or under the conditions that are used for the thermocycling DEXTAQ is 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 reduced ability to incorporate dideoxynucleotides compared to the said first thermostable DNA polymerase. The first DNA polymerase mainly produces shortened products that accumulate exponentially during the cycles and contribute to the sequence ladder that is generated whereas the second DNA polymerase, which has a reduced ability to incorporate dideoxynucleotides compared to the first said thermostable DNA polymerase, primarily produces products of complete length which accumulate exponentially and serve in subsequent cycles as a template for the production of further DNA strands of complete length as well as tem
Kilger Christian
Motz Michael
Horlick Kenneth R.
Roche Diagnostics GmbH
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