Many target and signal amplification methods have been described in the literature, but none are believed to offer the combination of high specificity, simplicity, and speed. General reviews of these methods have been prepared by Landegren, U., et al., Science 242:229-237 (1988) and Lewis, R., Genetic Engineering News 10:1, 54-55 (1990). These methods include polymerase chain reaction (PCR), PCR in situ, ligase amplification reaction (LAR), ligase hybridization, Q.beta. bacteriophage replicase, transcription-based amplification system (TAS), genomic amplification with transcript sequencing (GAWTS), nucleic acid sequence-based amplification (NASBA) and in situ hybridization. Some of these various techniques are described below.
Polymerase Chain Reaction (PCR)
PCR is the nucleic acid amplification method described in U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis. PCR consists of repeated cycles of DNA polymerase generated primer extension reactions. The target DNA is heat denatured and two oligonucleotides, which bracket the target sequence on opposite strands of the DNA to be amplified, are hybridized. These oligonucleotides become primers for use with DNA polymerase. The DNA is copied by primer extension to make a second copy of both strands. By repeating the cycle of heat denaturation, primer hybridization and extension, the target DNA can be amplified a million fold or more in about two to four hours. PCR is a molecular biology tool which must be used in conjunction with a detection technique to determine the results of amplification. The advantage of PCR is that it may increase sensitivity by amplifying the amount of target DNA by 1 million to 1 billion fold in approximately 4 hours. The disadvantage is that contamination may cause false positive results, or reduced specificity.
Transcription-based Amplification System (TAS)
TAS utilizes RNA transcription to amplify a DNA or RNA target and is described by Kwoh et al. (1989) Proc. Natl. Acad. Sci., USA 86:1173. TAS uses two phases of amplification. In phase 1, a duplex cDNA is formed containing an overhanging, single-stranded T7 transcription promoter by hybridizing a polynucleotide to the target. The DNA is copied by reverse transcriptase into a duplex form. The duplex is heat denatured and a primer is hybridized to the strand opposite that containing the T7 region. Using this primer, reverse transcriptase is again added to create a double stranded cDNA, which now has a double stranded (active) T7 polymerase binding site. T7 RNA polymerase transcribes the duplex to create a large quantity of single-stranded RNA.
In phase 2, the primer is hybridized to the new RNA and again converted to duplex cDNA. The duplex is heat denatured and the cycle is continued as before. The advantage of TAS over PCR, in which two copies of the target are generated during each cycle, is that between 10 and 100 copies of each target molecule are produced with each cycle. This means that 10.sup.6 fold amplification can be achieved in only 4 to 6 cycles. However, this number of amplification cycles requires approximately three to four hours for completion. The major disadvantage of TAS is that it requires numerous steps involving the addition of enzymes and heat denaturation.
Transcriptions Amplification (3SR)
In a modification of TAS, known as 3SR, enzymatic degradation of the RNA of the RNA/DNA heteroduplex is used instead of heat denaturation, as described by Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874. RNAse H and all other enzymes are added to the reaction and all steps occur at the same temperature and without further reagent additions. Following this process, amplifications of 10.sup.6 to 10.sup.9 have been achieved in one hour at 42.degree. C.
Ligation Amplification (LAR/LAS)
Ligation amplification reaction or ligation amplification system uses DNA ligase and four oligonucleotides, two per target strand. This technique is described by Wu, D. Y. and Wallace, R. B. (1989) Genomics 4:560. The oligonucleotides hybridize to adjacent sequences on the target DNA and are joined by the ligase. The reaction is heat denatured and the cycle repeated. LAR suffers from the fact that the ligases can join the oligonucleotides even when they are not hybridized to the target DNA. This results in a high background. In addition, LAR is not an efficient reaction and therefore requires approximately five hours for each cycle. Thus, the amplification requires several days for completion.
Q.beta. Replicase
In this technique, RNA replicase for the bacteriophage Q.beta., which replicates single-stranded RNA, is used to amplify the target DNA, as described by Lizardi et al. (1988) Bio/Technology 6:1197. First, the target DNA is hybridized to a primer including a T7 promoter and a Q.beta. 5' sequence region. Using this primer, reverse transcriptase generates a cDNA connecting the primer to its 5' end in the process. These two steps are similar to the TAS protocol. The resulting heteroduplex is heat denatured. Next, a second primer containing a Q.beta. 3' sequence region is used to initiate a second round of cDNA synthesis. This results in a double stranded DNA containing both 5' and 3' ends of the Q.beta. bacteriophage as well as an active T7 RNA polymerase binding site. T7 RNA polymerase then transcribes the double-stranded DNA into new RNA, which mimics the Q.beta.. After extensive washing to remove any unhybridized probe, the new RNA is eluted from the target and replicated by Q.beta. replicase. The latter reaction creates 10.sup.7 fold amplification in approximately 20 minutes. Significant background may be formed due to minute amounts of probe RNA that is non-specifically retained during the reaction.
Chiron Signal Amplification
The Chiron system, as described by Urdea et al. (1987) Gene 61:253, is extremely complex. It utilizes 12 capture oligonucleotide probes, 36 labeled oligonucleotides, 20 biotinylated immobilization probes that are crosslinked to 20 more enzyme-labeled probes. This massive conglomerate is built-up in a stepwise fashion requiring numerous washing and reagent addition steps. Amplification is limited because there is no cycle. The probes simply form a large network.
ImClone Signal Amplification
The ImClone technique utilizes a network concept similar to Chiron, but the approach is completely different. The ImClone technique is described in Kohlbert et al. (1989) Mol. and Cell Probes 3:59. ImClone first binds a single-stranded M13 phage DNA containing targeted probe. To this bound circular DNA is then hybridized about five additional DNA fragments that only bind to one end and the other end hangs freely out in the solution. Another probe set is then hybridized to the hanging portion of the previous set of probes. The latter set is either labeled directly with an enzyme or it is biotinylated. If it is biotinylated, then detection is via a streptavidin enzyme complex. In either case, detection is through an enzyme color reaction. Like the Chiron method, the ImClone method relies on build-up of a large network. Because there is no repeated cycle, the reaction is not geometrically expanded, resulting in limited amplification.
While the nucleic acid amplification methods described above allow for the detection of relatively small quantities of target nucleic acid molecules, there is a need for the ability to detect target nucleic acid molecules in a shorter amount of time with less background interference. Problems inherent in PCR and other amplification techniques involve sample contamination during the collection techniques and the presence of amplicons (amplified target DNA). There are problems with nonspecific target amplification mediated by closely related sequences and the production of primer dimers. There is also poor control of specificity, resulting in false positive reactions, and poor control of sensitivity, resulting in false negative reactions. PCR results must often be confirmed and validated by other techniques such as probe hybridization, Southern blotting or in situ hybridization.
Additionally, PCR and amplification techniques can only be used with very small amounts of starting sample DNA, in the range of a maximum of 1 microgram. This negates use of PCR techniques for the detection of low copy number nucleic acid targets. For example, early detection of HIV infection, soon after the initial viral infection, would be almost impossible to detect using PCR.
Thus, compositions, methods and kits are needed that are capable of detecting specific nucleic acid sequences and isolating them. Especially needed are methods and kits that would allow for the detection of low copy number nucleic acid target sequences. Additionally, there is need for methods and kits that provide the flexibility that would allow for isolation of nucleic acid sequences using a desired level of specificity.
What is also needed are methods that do not use amplification techniques, but do allow for the isolation of a specific target sequence from any amount of starting nucleic acid, especially large amounts, and have the flexibility to accomplish the isolation at several levels of specificity, depending on the level of specificity desired.
USPTO Backgrounds
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