Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-03-16
2001-07-17
Jones, W. Gary (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200
Reexamination Certificate
active
06261773
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to processes for nucleic acid amplification useful for biological research and clinical diagnosis, and reagents for use in the processes. More specifically, the present invention relates to processes for sequence-specific nucleic acid amplification which enable highly sensitive detection of nucleic acids, and reagents for use in the processes.
DESCRIPTION OF RELATED ART
In recent years, PCR (Polymerase Chain Reaction: Science, Vol. 239, pp. 487-491, 1988) and other techniques for artificially amplifying nucleic acids including DNA and RNA are widely known and utilized for medical and biological researches. The nucleic acid amplification techniques are indispensable for detection, cloning and modification of genes, and are finding wider application in clinical diagnosis. For example, a method for detecting pathogens with high sensitivity in a very short period has been developed and put into practical use, the method comprising amplifying a nucleic acid characteristic of a pathogen (e.g., Mycobacterium) and detecting the amplified nucleic acid. Also, application of the nucleic acid amplification techniques in detection of abnormalities of genes of humans and other animals is being researched for practical use.
In addition to PCR, a number of nucleic acid amplification techniques have been developed recently, which include LCR (Ligase Chain Reaction: Genomics 4, 560 (1989)), NASBA (Nucleic Acid Sequence-Based Amplification: Nature 350, p. 91 (1991)), SDA (Strand Displacement Amplification: Nucleic Acids Res. 20, 1961 (1992)), TMA (Transcription Mediated Amplification: J. Clin. Microbiol. 31, 3270 (1993)) and 3SR (Self Sustained Sequence Replication Reactions: Proc. Nat. Acad. Sci., USA 87, 1874-1878 (1990)).
Among these techniques, NASBA, TMA and 3SR are RNA amplification techniques wherein RNA is utilized as a target nucleic acid (template nucleic acid) for amplification.
The principle of these RNA amplification techniques is as follows. First, the target RNA is converted into a double-stranded DNA having a RNA promoter incorporated therein, using a DNA synthetase such as reverse transcriptase; and then 100 to 1,000 copies of RNA are obtained by transcription from the double-stranded DNA, using a RNA synthetase such as RNA polymerase. This cycle is repeated at a constant temperature to finally obtain several ten thousand to several million copies of the RNA.
Nucleic acid amplification techniques utilizing RNA as the target nucleic acid further include RT-PCR, which, however, involves the step of converting RNA into DNA by reverse transcription reaction, in advance of the PCR step. RT-PCR thus requires troublesome manipulations and much time (usually about 5 to 6 hours). In contrast, the other RNA amplification techniques mentioned above are advantageous in that they are simple and take less time since a series of reactions proceed in one operation.
Another future of these RNA amplification techniques is that the target RNA is amplified preferentially even if the sample contains DNA homologous to the target RNA. The RNA amplification techniques are therefore useful for, for example, precisely detecting or quantitating a desired RNA or the expression of a desired gene. On the other hand, amplification from contaminating DNA is inevitable in the PCR step of RT-PCR, resulting in a contaminated amplification product.
Further, these RNA amplification techniques, in particular NASBA, are characterized in that series of amplification reactions can be carried out at a constant temperature. Therefore, these techniques can be performed in a simple apparatus such as an ordinary constant temperature bath, instead of a thermal cycler.
Among these RNA amplification techniques, NASBA is characterized by high amplification efficiency. In an ideal reaction system of NASBA, the target RNA undergoes a rapid amplification reaction and gives a high positive signal if present beyond the detection limit, whereas it is not amplified and gives no signal if present below the detection limit. Accordingly, NASBA is beneficial in that it can give very clear positive
egative results.
Thus, these RNA amplification techniques are relatively advantageous from the viewpoints of operation simplicity and amplification efficiency.
However, nucleic acid amplification techniques, including the RNA amplification techniques and other techniques such as PCR, have a constant problem of non-specific amplification reactions. It is presumed that the non-specific amplification reactions occur mainly because primers will bind to non-target-nucleic acid sequences. Such reactions consume the primers and substrates (such as deoxyribonucleotide or ribonucleotide) and reduce the amplification efficiency of the target nucleic acid. Further, the presence of non-specific amplification reaction products in the amplification product decreases the detection signal from the amplified target nucleic acid and impairs the detection sensitivity.
For example, non-specific amplification reactions in PCR result in a reduced signal from the band corresponding to the desired amplification product and unclear electrophoresis profiles in the subsequent electrophoretic detection of the amplification product. Similar results are reached when the detection is carried out by other methods than electrophoresis, such as hybridization usually employed for NASBA product.
Occurrence of non-specific reactions greatly depends on properties of the primers used for amplification and selection of a sequence to be amplified. For preventing non-specific reactions, it is therefore necessary to select primers which are unlikely to cause non-specific reactions, taking into consideration the Tm value of the target nucleic acid and specificity and stereostructure of the sequence. However, selection of primers is often restricted since the sequence to be amplified is usually predetermined.
Studies have been made on a PCR method wherein non-specific reactions are reduced by controlling physical conditions such as temperature and period for annealing in each cycle and wherein the signal is improved by increasing the number of amplification cycles. Also reported is a two-step PCR method (nested PCR) wherein the detection sensitivity is improved by using a second pair of primers for synthesizing an internal region of the target sequence (J. Yourno et al., PCR Methods Applic. 2: 60 (1992)).
However, the former method is not applicable in NASBA and like techniques performed at a constant temperature, while the latter method requires complicated manipulations and thus greatly impairs the simpleness, an advantage of NASBA and like RNA amplification techniques.
SUMMARY OF THE INVENTION
An object of the invention is to provide a process for nucleic acid amplification which enables highly sensitive detection of a nucleic acid.
Another object of the invention is to provide an improved process for RNA amplification, specifically, an improved process for RNA amplification (in particular NASBA) wherein non-specific amplification reactions are significantly reduced to enable highly sensitive detection of a desired amplification product.
A further object of the invention is to provide a reagent for nucleic acid amplification for use in the above nucleic acid amplification process, preferably the improved process for RNA amplification, more preferably the improved process for NASBA.
A still further object of the invention is to provide a reagent kit useful for the above nucleic acid amplification process and for detection of the amplified nucleic acid.
The present inventors found that, when a specific compound such as ethylenediaminetetraacetic acid is added to a reaction mixture for nucleic acid amplification to carry out an amplification reaction in the presence of the compound, non-specific amplification reactions can be significantly inhibited, with the results that the desired amplification product can be detected as a high signal and that the detection sensitivity is markedly increased. The present invention has been accomplish
Kondo Motohiro
Segawa Masaya
Takarada Yutaka
Jones W. Gary
Kenyon & Kenyon
Toyo Boseki Kabushiki Kaisha
Tung Joyce
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