We have developed a novel, isothermal DNA amplification strategy that employs 29 DNA polymerase and rolling circle amplification to generate high-quality templates for DNA sequencing reactions. The TempliPhi DNA amplification kits take advantage of the fact that cloned DNA is typically obtained in circular vectors, which are readily replicated in vitro using 29 DNA polymerase by a rolling circle mechanism. This single subunit, proofreading DNA polymerase has excellent processivity and strand displacement properties for generation of multiple, tandem double-stranded copies of the circular DNA, generating as much as 107-fold amplification. Large amounts of product (13 g) can be obtained in as little as 4 hours. Input DNA can be as little as 0.01 ng of purified plasmid DNA, a single bacterial colony, or a 1 L of a saturated overnight culture. Additionally, the presence of an associated proofreading function within the 29 DNA polymerase ensures high-fidelity amplification. Once completed, the product DNA can be used directly in sequencing reactions. Additionally, the properties of 29 DNA polymerase and its use in applications such as amplification of human genomic DNA for genotyping studies is discussed.

We previously found that strand transfer by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is promoted at sites where RT pauses during synthesis. In this report, strand transfer is measured within the 5 transactivation response region (TAR) of HIV-1 RNA. We hypothesized that the stable hairpin structure of TAR would induce RT pausing, promoting RNase H-directed cleavage of the template and subsequent transfer at that site. We further predicted that HIV-1 nucleocapsid protein (NC), known to melt secondary structures, would decrease transfer. We show that TAR created a strong pause site for RT, but NC significantly promoted strand transfer. The effect of NC is specific, since other single strand binding proteins failed to stimulate transfer. In another unexpected outcome, preferred positions of internal transfer were not at the pause site but were in the upper stem and loop of TAR. Thus, we propose a new mechanism for transfer within TAR described by an interactive hairpin model, in which association between the donor and the acceptor templates within the TAR stem promotes transfer. The model is consistent with the observed stimulation of strand transfer by NC. The model is applicable to internal and replicative end transfer.

According to the current model for retroviral replication, strand displacement of the long terminal repeat (LTR) is a necessary step during plus strand DNA synthesis in vivo. We have investigated the ability of human immunodeficiency virus reverse transcriptase (HIV-RT) to synthesize in vitro over a 634-nucleotide HIV LTR DNA template, having or lacking a single full-length DNA downstream primer. The presence of the downstream primer resulted in an approximately 12-fold reduction in the rate of upstream primer elongation. Addition of Escherichia coli single-stranded binding protein (SSB) or human replication protein A (RP-A) enhanced strand displacement synthesis; however, addition of HIV nucleocapsid protein (NC) did not. The presence of excess single-stranded DNA complementary to the downstream primer did not stimulate displacement synthesis. Interestingly, we observed that the elongating upstream primer could readily transfer to this DNA. This observation suggests that recombination is favored during strand displacement synthesis in vivo.

No abstract

HIV-1 reverse transcriptase (RT) degrades the plus strand viral RNA genome while synthesizing the minus strand of DNA. Many RNA fragments, including the polypurine tracts, remain annealed to the new DNA. Several RTs are believed to bind after synthesis to degrade all RNA fragments except the polypurine tracts by a polymerization-independent mode of RNase H activity. For this latter process, we found that RT positions the RNase H active site approximately 18 nt from the 5 end of the RNA, making the primary cut. The enzyme rebinds or slides toward the 5 end of the RNA to make a secondary cut creating two products 8 -9 nt long. RT then binds the new 5 end of the RNA created by the first primary or the secondary cuts to make the next primary cut. In addition, we observed another type of RNase H cleavage specificity. RT aligns the RNase H active site to the 3 end of the RNA, cutting 5 residues in. We determined the relative rates of these cuts, defining their temporal order. Results show that the first primary cut is fastest, and the secondary and 5-nt cuts occur next at similar rates. The second primary cuts appear last. Based on these results, we present a model by which RT progressively cleaves RNA fragments.H IV is the causative agent of AIDS (1). To establish an infection, HIV must integrate a double-stranded viral DNA into the host chromosome (2). The virus-encoded reverse transcriptase (RT) converts the single-stranded viral RNA genome into that double-stranded DNA. RT uses several different catalytic activities in this process. The polymerase function catalyzes DNA synthesis on RNA and DNA templates (3). RT also has an RNase H active site that cleaves RNA when annealed to DNA. RNase H activity is required for a number of steps in viral replication, including formation of primers and primer strand transfers (4). An important role for the RNase H is the complete removal of the original genomic RNA during the synthesis of the double-stranded DNA (5, 6).The genomic viral RNA first is converted to an RNA͞DNA hybrid and then to double-stranded DNA. Synthesis of the second DNA strand necessitates complete removal of the original RNA (7,8). The RNA genome is cut into small segments during and after synthesis of the RNA͞DNA hybrid (9-11). Studies show that two different modes of RNase H activity are important for the removal of the RNA (5, 12, 13). The first mode is carried out by the same RT performing DNA synthesis. This is the polymerization-dependent mode of cleavage (9)(10)(11)(14)(15)(16)(17). We showed that the HIV-1 RT synthesizing DNA leaves RNA oligomers in its wake, many still bound to the extended DNA primer (17, 18). These residual RNAs occur because cleavages of the RNA template happen less frequently than nucleotide addition. Kati et al. (19) examined the rates of both catalytic activities of RT and found that the polymerization rate is 7-10 times faster than the RNase H rate. This result demonstrates that the two activities are uncoupled, such that several nucleotides are added in the time required fo...