Self-Sustained Sequence Replication

Abstract: T h e processes of DNA replication, DNA ligation, and RNA transcription have provided the basis of distinct and separate in vitro nucleic acid amplification strategies. The first described and the most widely utilized amplification protocol is the polymerase chain reaction (PCR).O,2) The PCR method consists of multiple cycles of oligonucleotide primer-directed DNA replication that are punctuated by short periods of elevated temperature (92-97~ to permit strand separation of newly synthesized DNA. The modifications, adaptations, and enhancements of the basic PCR protocol now number in the hundreds (reviewed partially in refs. 3 and 4) and have arisen primarily to meet the challenges of the numerous biological questions to which this valuable technology has been applied. More recently, the description of an amplification method based on cycles of oligonucleotide-targeted ligation, called the ligase chain reaction (LCR) (see review by Barany in this issue), has offered users the ability to amplify short (oligonucleotide-length) segments of DNA and simultaneously to monitor these amplified segments for the presence of mutations. (s-7) Similar to the PCR protocol, the LCR method includes thermocycling steps to permit the denaturing of newly ligated oligonucleotide duplexes so that these products can serve as templates for subsequent cycles of amplification. The requirement for multiple heatdenaturation steps by both the PCR and LCR amplification methods has prompted the rapid development of programmable thermocycling instruments and has hastened the isolation and purification of thermostable forms of DNA polymerase (8,9) and ligase. (7,10,11) These thermostable enzymes allow the amplification reactions to be carried out over many cycles of heating and cooling without debilitating loss of catalytic activities. The necessity of employing thermocycling as an essential part of an in vitro amplification strategy has been obviated by changing the product of the amplification process from double-stranded DNA to single-stranded RNA, thereby eliminating the heat denaturation step from the procedure. RNA transcription is a process employed by all cellular and viral systems to copy discrete segments of nucleotide sequences into multiple single-stranded RNA molecules. In exploring modifications of the previously described in vitro transcription-based amplification system (TAS), (12,13) it was discovered that isothermal replication of a targeted nucleic acid is possible using a concerted three-enzyme, in vitro reaction. This amplification strategy has been termed a selfsustained sequence replication (3SR) reaction, and was modeled after the general scheme employed during retroviral replication. (~4) In this reaction, activities of avian myeloblastosis virus (AMV) reverse transcriptase (RT), Escherichia coli RNase H, and T7 RNA polymerase produce an average 10-fold amplification every 2.5 min (for the first 15 min), leading to 107-fold amplification in 60 min. (14) Figure 1 provides a description of the steps in the 3SR amplification reaction. This report discusses additional developments in the originally described 3SR reaction. (14) Such developments include: (1) optimization of substrate concentrations, (2) the effect of temperature, pH, and ionic strength on amplification productivity, and (3) the reaction conditions required that enable the 3SR reaction to be carried out with only AMV RT and T7 RNA polymerase (two-enzyme 3SR reaction).

Abstract: The present disclosure provides details of an invention comprising an amplification system for the detection of target nucleic acids, most particularly target nucleic acid sequences, in an isothermal setting wherein all of the reagents necessary to conduct the amplification are present and the reactions are self-sustaining and continuous. Featured is a selective enzymatic digestion of a RNA/DNA duplex, formed by hybridization of a DNA primer with target nucleic acid, thus freeing the DNA strand for further hybridization followed by primer extension to provide a DNA duplex that can serve as a template for production of a plurality of transcripts that can be recycled and/or detected as such for deduced presence of target nucleic acid sequence.

Abstract: A nucleic acid sequence-based amplification (NASBA) technique for the detection of hepatitis A virus (HAV) in foods was developed and compared to the traditional reverse transcription (RT)-PCR technique Oligonucleotide primers targeting the VP1 and VP2 genes encoding the major HAV capsid proteins were used for the amplification of viral RNA in an isothermal process resulting in the accumulation of RNA amplicons Amplicons were detected by hybridization with a digoxigenin-labeled oligonucleotide probe in a dot blot assay format Using the NASBA, as little as 04 ng of target RNA/ml was detected per comparison to 4 ng/ml for RT-PCR When crude HAV viral lysate was used, a detection limit of 2 PFU (4 x 10(2) PFU/ml) was obtained with NASBA, compared to 50 PFU (1 x 10(4) PFU/ml) obtained with RT-PCR No interference was encountered in the amplification of HAV RNA in the presence of excess nontarget RNA or DNA The NASBA system successfully detected HAV recovered from experimentally inoculated samples of waste water, lettuce, and blueberries Compared to RT-PCR and other amplification techniques, the NASBA system offers several advantages in terms of sensitivity, rapidity, and simplicity This technique should be readily adaptable for detection of other RNA viruses in both foods and clinical samples

Abstract: Mutations at amino acid positions 67, 70, 215, and 219 in the human immunodeficiency virus type 1 (HIV-1) pol gene correlate with the emergence of resistance to zidovudine (AZT). These four positions were monitored in viral RNA extracted from infected peripheral blood mononuclear cells (PBMC) and viral stocks obtained after coculture with uninfected lymphocytes. Genotype determinations were made using the self-sustained sequence replication (3SR) and differential bead-based sandwich hybridization (BBSH) assay. The hybridization results obtained by 3SR and BBSH analyses were verified by dideoxynucleotide sequencing of the 3SR products. Correlation of 3SR and BBSH with polymerase chain reaction and Southern hybridization analyses of the PBMC and corresponding viral isolates indicated that PBMC and corresponding HIV-1 isolates may differ in their genotypes at the monitored amino acid positions, variations from the wild-type nucleotide sequence may occur proximal to the codons being monitored, and viral isolates possessing the same genotypes at the four monitored amino acid positions showed a threefold variation in their ID50 measurements.