Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-12-12
2002-08-06
Benzion, Gary (Department: 1637)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C422S198000, C422S198000, C422S068100, C422S051000, C422S081000, C435S290400, C435S317100
Reexamination Certificate
active
06428987
ABSTRACT:
FIELD OF THE INVENTION
The invention concerns instruments for fast, selective replication of deoxyribonucleic acid (DNA) from biomaterial by the well-known polymerase chain reaction (PCR), working in individual duplication thermocycles.
DESCRIPTION OF THE RELATED ART
It is becoming more and more important for the medical care of patients that analysis methods in genetic engineering are made available which work very quickly. One example of this is the identification of infectious microorganisms, which still requires days at present, but actually requires treatment at the earliest possible stage, in the initial hours if possible. More intense will be the demand for quick analysis during examinations of tissue possibly affected by cancer or other disease during surgery on the open patient by means of oncogenetic, virological or bacteriological analyses. Here, a maximum analysis time of about ten minutes is required.
Mass spectrometry today provides very fast, highly sensitive analysis methods for the size of amplified DNA segments. Advances in matrix-assisted laser desorption and ionization (MALDI) make it possible to analyze about 20 samples including the MALDI preparation, the introduction of DNA MALDI samples into the mass spectrometer, the MALDI analysis and the data evaluation up to presentation on the screen in less than three minutes. The tissue cells and DNA extraction can be lysed in less than two minutes.
This maximum of five minutes total for sample preparation and mass spectrometry analysis stands in contrast to times of three hours for classic PCR replication. Extreme reductions in these times are on the horizon however. In one instrument, available commercially in the meantime, this time has already been reduced to about 20 minutes. In a recent publication (A. T. Woolley et al., “Functional Integration of PCR Amplification and Capillary Electrophoresis in a Microfabricated DNA Analysis Device”, Anal. Chem. 68, 4081, December 1996), DNA in 20 microliters of reaction solution was amplified through 30 cycles in only 15 minutes in a miniature chamber made of polypropylene. Even this time is, however, too long for a fast analysis in the above sense. The goal must be to perform the PCR amplification in only two to three minutes.
As is known, DNA consists of two complementary chains made up of four nucleotides, the sequence of which forms the genetic code. Each nucleotide consists of a sugar (ribose), a phosphoric acid group and a base. Two bases each are complementary to one another. Sugar and phosphoric acid form the continuous chain of the DNA (or the so-called backbone), the four characteristic bases are each lateral branches attached to the sugar. Both complementary chains or single strands of DNA are coiled around one another in the form of a double helix, whereby two complementary nucleotides each are connected to one another via hydrogen bridges between the bases and thus form a so-called double strand.
The basis for many analysis methods in genetics is the selectively functioning PCR (polymerase chain reaction), a simple replication method for specifically selected DNA pieces in a test tube, first developed in 1983 by K. B. Mullis (who received the Nobel Prize for this in 1993) and which, after the introduction of temperature stable polymerases, went on to unequalled success in genetic engineering laboratories.
PCR is the specific replication of a relatively short segment of double-stranded DNA, precisely sought from the genome, in simple temperature cycles. Selection of the DNA segment is through a so-called pair of primers, two DNA pieces with about 20 bases length apiece, which (described somewhat briefly and simply) encode the bilateral ends of the selected DNA segment. Replication is performed by an enzyme called polymerase, which represents a chemical factory in a molecule. The PCR reaction takes place in aqueous solution in which a few molecules of the original DNA and sufficient quantities of polymerase, primers, triphosphates of the four nucleic acids (so-called “substrates”), activators and stabilizers are present. In every thermal cycle, the DNA double helix is first “melted” at about 95° C., whereby both strands are separated from one another. At about 55° C., the primers are then attached to complementary nucleotide sequences of the DNA single strands (“hybridization”). At 72° C. the double helixes are reconstructed by elongation of the primers, done by the temperature-resistant polymerase (e.g. taq-polymerase). Complementary nucleotides are bonded, one after the other, to a specific end of the primers to form two new double helixes. In this way, the selected DNA segment is duplicated in principle between the primers. Therefore, over 30 cycles, around one billion DNA segments are generated from one single double-strand of DNA as original material. (In a more exact description, the shortening to the DNA segment between the primers only occurs statistically with further replications).
The duration of time for a thermal cycle is practically only dependent on the rate of heating up and cooling down, which is subsequently dependent upon the volume of liquid, the dimensions of the chamber and the thermal conductivity of the chamber walls and the reaction solution. For every thermal stage, only a few seconds are necessary in principle, sometimes even less.
In the above cited article by Woolley et al., in which the PCR amplification for 30 cycles only lasted 15 minutes, the following times were required, for example, for the work in the three thermal stages: 2 seconds at 96° C. for melting, 5 seconds at 55° C. for the primer attachment and 2 seconds at 72° C. for reconstruction. The remaining time of 21 seconds per cycle was used for the thermal transitions. The DNA melts almost instantaneously at a temperature a few degrees above the “melting temperature.” Analyses have shown that heating to this temperature for one half second suffices for complete separation of all double helix structures. Precise maintenance of the temperature is not even especially critical here, as long as one remains above the melting temperature but below a coagulation temperature. Hybridization also does not need much time if the primers are available in sufficient concentration. At an optimal concentration, about one to two seconds are enough. For hybridization, the temperature is even less critical; it need only remain under 60° C. to proceed sufficiently fast. Optimal conditions are at about 55° C.
The growth of the attached primers into a complementary DNA molecule through the polymerase, known as “reconstruction” in the following, has a very high velocity. 500 to 1,000 bases can be bonded per second under optimal thermal and concentration conditions by the polymerase. Since generally only DNA segments of a maximum of 400 bases in length are necessary for the analyses, two seconds are quite sufficient for this reconstruction phase. For this process of reconstruction of a new double helix, good maintenance of the optimal temperature is required in order to achieve the high rate of reconstruction.
Theoretically, a PCR reaction cycle could thus be concluded in less than 5 seconds, under the precondition that heat can be introduced or removed up to each sufficient thermal equilibrium in about ¼ second each. One such ideal thermal curve for a PCR cycle is shown in FIG.
1
. The introduction and removal of heat are the critical time-determining variables here.
By the addition of only one primer pair, uniform DNA segments can be replicated. However, if several different primer pairs are added at the same time, several DNA segments will also be replicated at the same time (“multiplexed PCR”). This type of multiplexed PCR is frequently used and often has special advantages. For so-called “fingerprinting” for the identification of individuals through DNA segments of variable length (methods of “VNTR=Variable Number of Tandem Repeats” or “AMP−FLP=Amplified Fragment Length Polymorphism”), it makes the analyses faster. Here through the selection of primers, which
Benzion Gary
Bruker Daltonik GmbH
Tung Joyce
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