Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1998-04-06
2000-03-07
Horlick, Kenneth R.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435 6, 435 911, 435 9121, 536 231, 536 243, 536 2431, C12P 1934, C12Q 168, C07H 2104
Patent
active
060338814
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
(i) Field of Invention
The present invention relates to the field of molecular biology and recombinant DNA-technology (genetic engineering). The invention allows a simplified in vitro synthesis of nucleic acids. The herein described methods are especially applicable to the amplification of nucleic acids with primer-dependent DNA/RNA-polymerases, DNA- and RNA-ligases. The invention has numerous applications in molecular biology, medical diagnostics, environmental and forensic analysis.
The invention relates especially to a process for isothermal, non-transcription based, amplification of nucleic acids and nucleic acid sequences respectively, by means of enzymes whereby the nucleic acids and nucleic acid sequences respectively, may be at least partially separated into single strands and/or transcribed.
The amplification proceeds by enzymatic incorporation of at least two oligonucleotide building blocks with a sequence essentially complementary to the sequence of the ends of the complementary strands of the nucleic acid or nucleic acid sequence to be amplified, wherein the product of the building blocks itself corresponds to the nucleic acid and nucleic acid sequence to be amplified.
(ii) Description of Related Art
The in vitro-amplification of nucleic acids, such as DNA and RNA and nucleic acid sequences, respectively, has many applications and may be performed in different ways (amplification in this document means a substantial amplification, i.e. more than doubling of the starting material).
The principle of amplification of DNA by means of polymerase activity has been described in detail by Kleppe et al., J. Mol. Biol. 56: 341 (1971). Accordingly, oligonucleotides are used as starting points for enzymatic DNA-synthesis such that the product of this synthesis may be used as template for further synthesis. According to this principle the complementary nucleic acids strands have to be separated repeatedly between the single steps of the synthesis. U.S. Pat. No. 4,683,202 describes the realization of this principle.
Regarding the applied temperature one may distinguish especially two processes: processes with cycling temperature changes between amplification temperature and denaturing (strand separation) temperature, wherein the nucleic acids or the nucleic acid sequences prior to the amplification step have to be separated essentially completely into single strands. The other amplification processes work isothermally.
The polymerase chain reaction (PCR), for example, belongs to the first group of temperature cycling processes, wherein a nucleic acid sequence can be amplified exponentially in such a reaction where the temperature is subjected to a cyclic change. For such an amplification, usually a thermostable polymerase is used [e.g. Saiki et al., Science, 230, 1350-1354 (1985), Saiki et al., Science, 239, 487 (1988), respectively].
According to U.S. Pat. No. 4,683,195 (Mullis et al.) one can perform in a temperature cycling process the strand separation following the amplification step by any suitable denaturation method, it may be chemically, physically or enzymatically. In the referred document physical strand separation methods are preferred, e.g. heating the nucleic acid until complete denaturation (>99%), i.e. separated into two single strands. A typical heat denaturation is performed at temperatures between 90.degree. C. and 105.degree. C. and lasts generally between 0.5 and 3 minutes. Preferred temperatures are between 90.degree. C. and 100.degree. C. for 0.5 to 3 minutes. When using a thermolabile enzyme (U.S. Pat. No. 4,683,202) one has to add fresh enzyme after each strand separation by heat. By using a thermostable enzyme one does not need to interrupt the thermocycling for enzyme addition. The process is performed by means of temperature cycling apparatus "simultaneously".
A further process in the group of thermocycling processes is the so called ligase chain reaction (LCR) e.g. according to F. Barany in Proc. Natl. Acad. Sci. USA, 88, 189-193 (1991), wherein th
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The Journal of Biological Chemistry, M. Iyer et al., "Accelerated Hybridization of Oligonucleotides to Duplex DNA", vol. 270, No. 24, Issue of Jun. 16, pp. 14712-14717, 1995.
The Journal of Biological Chemistry, E. Kmiec, et al., "DNA Strand Exchange in the Absence of Homologous Pairing", vol. 269, No. 13, Issue of Apr. 1, pp. 10163-10168, 1994.
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Himmler Gottfried
Schlederer Thomas
Horlick Kenneth R.
Siew Jeffrey
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