Fergal Hill

3-Nitropyrrole and 5-nitroindole as universal bases in primers for DNA sequencing and PCR

3-Nitropyrrole and 5-nitroindole have been assessed as universal bases in primers for dideoxy DNA sequencing and in the polymerase chain reaction (PCR). In contrast to a previous report, we have found that the introduction of more than one 3-nitropyrrole residue at dispersed positions into primers significantly reduced their efficiency in PCR and sequencing reactions. Primers containing 5-nitroindole at multiple dispersed positions were similarly affected; for both bases only a small number of substitutions were tolerated. In PCR experiments neither base, when incorporated into primers in codon third positions, was as effective as hypoxanthine, which was incorporated in six codon third positions in a 20mer oligomer. However, primers containing up to four consecutive 5-nitroindole substitutions performed well in both PCR and sequencing reactions. Consecutive 3-nitropyrrole substitutions were tolerated, but less well in comparable reactions.

Nitroindoles as Universal Bases

Abstract Nitroindoles behave as non-discriminatory bases when incorporated into oligodeoxynucleotides, without causing significant destabilisation of the duplex, and exhibit higher melting temperatures than 3-nitropyrrole. When 5-nitroindole and 3-nitropyrrole are incorporated into oligomers for use as primers for sequencing and the polymerase chain reaction, both give products but are not as effective as hypoxanthine.

EASEL, A GYPSY LTR-RETROTRANSPOSON IN THE SALMONIDAE

Despite the close similarities between retroviruses and the gypsy/Ty3 group of LTR-retrotransposons their host ranges are largely distinct: the retroviruses are found only in vertebrates, whereas the gypsy LTR-retrotransposons are almost exclusively restricted to invertebrates, plants and fungi. Here we report the amplification by PCR, and characterisation, of one of the first LTR-retrotransposons to be discovered in vertebrates - in several members of the piscine family Salmonidae. Phylogenetic analysis of this retroelement, termed easel, indicates that it is probably a phylogeneticaly basal member of the gypsy group of LTR-retrotransposons and occurs in some of the same species from which retroviruses have previously been isolated. Thus some members of the Salmonidae are the first organisms known to harbour both retroviral branch elements and the gypsy LTR-retrotransposon branch elements. This creates an overlap in the host ranges of the two retroelement families.

Enzymatic Recognition of Acyclic Universal Base Analogues in Oligonucleotides

Abstract The effects of the acyclic analogues 3 and 4 on recognition by commonly used enzymes in recombinant DNA work have been investigated. This report shows that acyclic sugars can function in place of the normal 2′- deoxyribose moiety with some enzymes, expanding the range of possible manipulations.

5′-Tailed Octanucleotide Primers for Cycle Sequencing

Abstract Recently we reported the use of octamer primers tailed with 5-nitroindole for use as primers in cycle sequencing reactions. Here we report the successful use of some other universal base analogues to improve the effectiveness of an octamer sequencing primer. These analogues are 5-nitroindazole, 3-nitropyrrole and benzimidazole.

USE OF KARPAS HIV CELL TEST TO DETECT ANTIBODIES TO HIV-2

Commercial enzyme-linked immunosorbent assays for HIV-1 infection often fail to detect antibodies to HIV-2. A simplified version of the HIV cell test detects antibodies to this new virus and enables typing of HIV infections. Since sera from 3 macaque monkeys infected with simian immunodeficiency virus gave weakly positive reactions, this test holds promise for discovering any further strains of HIV that may exist or evolve.

Properties of Oligonucleotides Containing the Transition Bases P and K

Abstract The properties of the degenerate nucleosides dP and dK, in templates and primers were determined. dP was copied as either pyrimidine, dK as either purine. In primers, an equimolar mixture of the two nucleosides functioned as a universal base equivalent in both sequencing and the polymerase chain reactions. Cloning of DNA containing dP or dK produced transition mutations in vivo.

Synthesis and Polymerase Incorporation Properties of a Tricyclic Pyrrolopyrimidine Related to N6-Hydroxy-2′-deoxyadenosine

Abstract The tricyclic nucleotide dLTP has been synthesised. It is incorporated into DNA using a variety of polymerases opposite both template T and dC in vitro and in vivo.

Random Mutagenesis Using 2-Amino-9-(2-Deoxy-β-D-Ribofuranosyl)Purine-5′-Triphosphate and the Polymerase Chain Reaction

Abstract Base analogues offer an attractive method for mutagenising DNA in combination with the polymerase chain reaction (PCR). We have synthesised the 5′-triphosphate-2′-deoxyribosyl derivative of 2-aminopurine (dAPTP), one of the first base analogues to be used for mutagenesis, and examined its utility in PCRs. An E. coli amber suppressor gene, supF, was used as a template for mutagenesis. The analogue induced exclusively transition mutations, but at a low frequency, consistent with its weak mutagenicity in vivo.