Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-03-06
2001-10-16
Zitomer, Stephanie W. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S403000, C536S023100, C536S024330, C536S025320, C436S173000
Reexamination Certificate
active
06303298
ABSTRACT:
The invention referres to a method for the preparation and selective replication of deoxyribonucleic acid (DNA) from biomaterial using PCR (polymerase chain reaction) for the analysis in time-of-flight mass spectrometers TOF) with matrix-assisted laser desorption and ionization (MALDI) for determining specific genetic features in biomaterial.
The invention consists, in a first step, of replicating the selected DNA segments by the PCR method in the usual unmodified fashion, and in a further enzymatic replication process, to replicate the DNA segments using modified substrates (the nucleic acids used for PCR) and specially prepared primers to such DNA analogs as are especially suitable for ionization by MALDI. Preparation of the primers particularly consists of introducing a charged group, which improves the ionization, and modification of the substrates is used to neutralize the negative charge on the phosphoric acid group of the DNA backbone. It is especially practical to immobilize the DNA on a surface for the final enzymatic reduplication process, for example on a prepared MALDI layer located on magnetic beads. In the final reduplication process, extremely little reagent is used, thus it is also possible in terms of low cost to use isotope-enriched material here in order to increase the mass resolving power.
PRIOR ART
“DNA fingerprinting” or “DNA typing” is currently revolutionizing the individual identification and characterization of hereditary factors (including factors for disease) in humans, animals, and plants. It is ultimately based on the determination of the molecular weight of replicated, highly variable DNA segments (“hypervariable DNA regions”, “polymorphic DNA segments”), such as so-called microsatellites (also known as STR=short tandem repeats), which consist of a varying number of repetitions of short sequences of two to five nucleotides, but also of other polymorphic forms, such as those of 2-allelic polymorphisms based on point mutations. The sizes of the amplified DNA segments are currently still measured by the slow and not completely automatable method of polyacrylamide gel electrophoresis (PAGE). All of these types of genetic typing could however be approached considerably better and more exactly using mass spectrometric measurements of the molecular weights. For the latter type of polymorphisms, a particular type of sample preparation using a mutation-dependent slicing of the DNA is required.
Medicine today is aware of far more than 3,000 monogenous diseases which can be teed back to such mutated changes of individual genes, while the still more widespread polygenous diseases have not yet been researched to any extent It can be expected that we will have identified all genes in five years, all mutations in ten years. Today, about 10,000 microsatelites are already known for humans, and it can be expected that there are more than ten times as many.
It is known that DNA consists of two complementary chains of four alternating 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 DNA (or the so-called backbone), the four characteristic bases are respective lateral branches attached to the sugar group. Both complementary chains of DNA are joined in the form of a double helix, whereby two complementary nucleotides are joined to one another via hydrogen bridges between the bases.
The basis for the fingerprint analysis method is the PCR (“polymerase chain reaction”), a simple in-vitro replication method for specifically selectable DNA pieces, first developed in 1983 by K. B. Mullis (Nobel Prize 1993), which, after the introduction of thermally stable polymerases, made an unprecedented conquest of genetic laboratories. The gel-electrophoretic analyses of minimal initial amounts of DNA, which have become feasible today due to this method, are performed on a PAGE basis (PAGE=polyacrylamide gel electrophoresis). However, specialists see no future in this type of analysis since it consistently eludes an often-attempted automation due to various types of frequently occurring artefacts and therefore requires a great number of personnel. In addition, it is slow and also quite expensive, due to its relatively heavy use of expensive reagents. This method was a pioneering procedure, though it has proven to be a bottleneck for further broadening of the method.
One method which shows promise on many levels is capillary electrophoresis, with a multiple of capillaries, though it only just starts to market with industrially produced commercial units.
PCR is the targeted replication of a precisely defined piece of double-stranded DNA. The DNA segment is selected by a so-called pair of primers, two DNA pieces with about 20 bases length apiece, which (described somewhat briefly and simply) encode both ends of the selected DNA piece. Replication is performed by an enzyme called polymerase, which represents a chemical factory in a molecule, in a simple thermal cycle. The PCR reaction takes place in aqueous solution in which a few molecules of the original DNA and sufficient quantities of polymerase, primers, nucleic acids and stabilizers are present The thermal cycles consist for example in melting of the double helix at 92° C., hybridization of the primer at 55° C., and reconstitution of the primers by prolongation to form the missing complement of the double helix through attachment of substrates by the polymerase at 72° C. In every thermal cycle, the amount of the selected DNA segments thus is doubled in principle. 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, both primers hybridize on the two different strands of the DNA and the shortening to the DNA segment between the primers only occurs statistically during further replication).
Under optimal conditions, the polymerase can attach about 500 to 1,000 bases per second to the primer. If there is a sufficient surplus of primers, polymerase and nucleotide substrates, the cycle period is practically only dependent on the rate of heating up and cooling down, which by itself depends upon the volume of liquid, the volume of the container, the thermal conductivity, and so on. For every temperature level only a few seconds are necessary in principle.
The primers are normally a part of the replicated DNA segments. Therefore chemical groups, which may be used for later detection (such as fluorescent groups), can be attached to the artificially produced primers.
By use 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 are replicated in the same PCR process (“multiplexed PCR”).
This type of multiplexed PCR is frequently used and often has special advantages. For so-called “fingerprinting”, for identification of individuals through DNA segments of variable length (method of “VNTR=Variable Number of Tandem Repeats” or “AMP-FLP=Amplified Fragment Length Polymorphism”), this makes the analyses faster. By selection of the primers which determine the average molecular weight of the DNA segments, the variations in molecular weight of DNA segments formed by the various primer pairs can be tailored to never or only seldomly overlap. This type of multiplex PCR requires an analyzer capable of simultaneous measurement of a large range of molecular weights.
The method has particular advantages in the identification of infectious organisms, since 20 types of bacteria (or viruses, yeasts, molds) can be detected at the same time, for example, with a single PCR replication procedure.
It must be expected that mass spectrometric measurements of molecular weight will be possible with much greater sensitivity and much higher velocities than with gel electrophoresis. The currently rapid progress in MALDI technology is leading t
Franzen Jochen
Gut Ivo Glynne
Bruker Daltonik GmbH
Tung Joyce
Zitomer Stephanie W.
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