Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1994-11-18
1997-03-04
Horlick, Kenneth R.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 911, 435 912, 435 9152, 436 94, C12Q 168, C12P 1934, G01N 3348
Patent
active
056078323
DESCRIPTION:
BRIEF SUMMARY
This invention relates to processes for the treatment of nucleic acid material in order to effect a complete or partial change from double stranded form to single stranded form and to processes of amplifying or detecting nucleic acids involving such denaturation processes.
BACKGROUND OF THE INVENTION
Double stranded DNA (deoxyribonucleic acid) and DNA/RNA (ribonucleic acid) and RNA/RNA complexes in the familiar double helical configuration are stable molecules that, in vitro, require aggressive conditions to separate the complementary strands of the nucleic acid. Known methods that are commonly employed for strand separation require the use of high temperatures of at least 60.degree. celsius and often 100.degree. celsius for extended periods of ten minutes or more or use an alkaline pH of 11 or higher. Other methods include the use of helicase enzymes such as Rep protein of E. coli that can catalyse the unwinding of the DNA in an unknown way, or binding proteins such as 32-protein of E.coli phage T4 that act to stabilise the single stranded form of DNA. The denatured single stranded DNA produced by the known processes of heat or alkali is used commonly for hybridisation studies or is subjected to amplification cycles.
U.S. Pat. No. 4,683,202 (Kary B Mullis et al, assigned to Cetus Corporation) discloses a process for amplifying and detecting a target nucleic acid sequence contained in a nucleic acid or mixture thereof by separating the complementary strands of the nucleic acid, hybridising with specific oligonucleotide primers, extending the primers with a polymerase to form complementary primer extension products and then using those extension products for the further synthesis of the desired nucleic acid sequence by allowing hybridisation with the specific oligonucleotides primers to take place again. The process can be carried out repetitively to generate large quantities of the required nucleic acid sequence from even a single molecule of the starting material. Separation of the complementary strands of the nucleic acid is achieved preferably by thermal denaturation in successive cycles, since only the thermal process offers simple reversibility of the denaturation process to reform the double stranded nucleic acid, in order to continue the amplification cycle. However the need for thermal cycling of the reaction mixture limits the speed at which the multiplication process can be carried out owing to the slowness of typical heating and cooling systems. It also requires the use of special heat resistant polymerase enzymes from thermophilic organisms for the primer extension step if the continuous addition of heat labile enzyme is to be avoided. It limits the design of new diagnostic formats that use the amplification process because heat is difficult to apply in selective regions of a diagnostic device and it also can be destructive to the structure of the DNA itself because the phosphodiester bonds may be broken at high temperatures leading to a collection of broken single strands. It is generally believed that the thermophilic polymerases in use today have a lower fidelity ie. make more errors in copying DNA than do enzymes from mesophiles. It is also the case that thermophilic enzymes such as TAQ polymerase have a lower turnover number than heat labile enzymes such as the Klenow polymerase from E.coli. In addition, the need to heat to high temperatures, usually 90.degree. celsius or higher to denature the nucleic acid leads to complications when small volumes are used as the evaporation of the liquid is difficult to control. These limitations have so far placed some restrictions on the use of the Mullis et al process in applications requiring very low reagent volumes to provide reagent economy, in applications where the greatest accuracy of copy is required such as in the Human Genome sequencing project and in the routine diagnostics industry where reagent economy, the design of the assay format and the speed of the DNA denaturation/renaturation process are important.
Denaturation/renaturation cyc
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Archer Patricia L.
Stanley Christopher J.
Horlick Kenneth R.
Scientific Generics Limited
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