Primer Binding Region Research Articles

Microchip and capillary electrophoresis for quantitative analysis of hepatitis C virus based on RT-competitive PCR

A method to quantitatively perform reverse transcription-competitive PCR (RT-cPCR) of hepatitis C virus followed by both microchip and capillary electrophoretic separation and detection was described. In this method, HCV wild-type (WT) RNA extracted from serum was coretrotranscribed and coamplified with a constant amount of recombinant internal standard (IS) RNA which had the same primer binding region as the target RNA and was constructed by removing a centrally located 25-bp segment from the target template. A linear calibration curve was constructed by adding IS RNA at a constant concentration of 8000 copies μl −1 into a series of RNA target standards ranging from 400 to 10 6 copies μl −1. The amplified IS and target DNA were detected by both capillary and microchip electrophoresis via laser-induced fluorescence (LIF) using Cy5-labelled primer as the fluorescence probe. The method was further demonstrated for the quantitation of clinical patients with low, medium, and high viral titer and the results were found to be comparable to those determined by the commercial bDNA assay.

In VitroCharacterization of Late Steps of RNA Recombination in Turnip Crinkle Virus: II. The Role of the Priming Stem and Flanking Sequences

Turnip crinkle carmovirus (TCV) has a uniquely high recombination frequency and nonrandom cross-over site distribution among the recombining TCV-associated satellite RNAs. Anin vitrosystem has been developed that includes a partially purified TCV replicase preparation (RdRp) and chimeric RNAs that resemble the putativein vivorecombination intermediates (Nagy, P. D., Zhang, C., and Simon, A. E.,EMBO J.17, 2392–2403, 1998). This system mimics the strand transfer and primer extension steps of recombination events. We characterize in detail three RNA factors that, in addition to the previously characterized motif1-hairpin, can influence the efficient generation of 3′-terminal extension products: (i) a primer binding region, termed the priming stem; (ii) a spacer region; and (iii) a U-rich sequence located 5′ of the motif1-hairpin. The priming stem is formed between the acceptor RNA and the nascent RNA synthesized from the donor RNA template in the recombinants. The stability and location of the priming stem relative to the motif1-hairpin can influence both the efficiency and initiation site of 3′-terminal extension. A short flexible spacer region between the motif1-hairpin and the priming stem can increase the efficiency of 3′-terminal extensions. A U-rich sequence 5′ of the motif1-hairpin facilitates 3′-terminal extensions and its function partly overlaps with that of the spacer region. These RNA factors may also affect the late steps of RNA recombination in TCV.

Nigerian rotavirus serotype G8 could not be typed by PCR due to nucleotide mutation at the 3' end of the primer binding site.

A rotavirus strain HMG89 from Nigeria with short electrophoretic pattern was typed G3 by PCR. A cDNA clone from the PCR product which hybridised in Northern blots to RNA segment 9 of the homologous Nigerian rotavirus strain HMG89 and laboratory reference strain 69M but not to other mammalian group A rotaviruses was sequenced. The VP7 gene 9 sequence is 1060 nucleotides long with two base deletions at positions 1034-1035. Sequence analysis of the primer (aAT8) used in the previous PCR serotyping assay revealed a mutation in one of the three nucleotide bases at the 3' end of the primer binding site accounting for our inability to serotype G8 strains in our samples. These findings demonstrate that PCR analysis can, albeit infrequently, lead to error in typing of rotaviruses due to small numbers of mutations in the primer binding region.

PCR typing of DNA fragments of the two short tandem repeat (STR) systems upstream of the human myelin basic protein (MBP) gene in Danes and Greenland Eskimos

DNA from the double short tandem repeat (STR) system MBP (locus 18q23-pter) was amplified by the polymerase chain reaction (PCR) and the two polymorphic repeat systems were separated by cutting with the restriction enzyme NlaIII. The lengths of the DNA fragments of the two MBP STR systems MBP-A and MBP-B were analyzed by vertical electrophoresis in polyacrylamide gels followed by silver staining. DNA samples from 112 unrelated Danes, 140 unrelated Greenland Eskimos, and 88 Danish mother/child pairs were analyzed. The distributions of MBP phenotypes were in Hardy-Weinberg equilibrium in both the Eskimo and Danish populations. Significant differences were observed between the distribution of fragments (‘alleles’) in Greenland Eskimos and in Danes. The allele MBP-A7 was considerably more frequent in Eskimos (0.2214) than in Danes (0.0775) and also the allele MBP-B9 was considerably more frequent in Eskimos (0.225) than in Danes (0.06). Strong gametic associations were found between fragments from the MBP-A and MBP-B series in both Danes and Eskimos. Some of the associations were different in Danes and Eskimos. In the 88 Danish mother/child pairs, the segregation of the MBP genotypes were in accordance with a genetic model of co-dominant inheritance and no mutation was found. Two MBP STR regions with irregular structures were sequenced. One fragment had a single base G to A transition at position 124 in the primer binding region between the MBP-A and MBP-B regions. In the other fragment, a deletion starting at position 117 and including the primer binding region between the MBP-A and MBP-B regions was found.

A region of primer binding variation at the D6S265 locus associated with HLA-A25 and HLA-A26 antigens.

D6S265 is a polymorphic dinucleotide repeat, mapped within 70 kb centromeric of HLA-A, on chromosome 6p21.3. While genotyping families for genetic linkage analysis, allele non-amplification resulting in apparent non-Mendelian inheritance was observed at the D6S265 locus in 15 individuals, on chromosomes carrying the HLA-A25 and HLA-A26 antigens. The D6S265 locus was sequenced in a variant individual homozygous for allele non-amplification, and in a non-HLA-A25/-A26 individual, homozygous for D6S265 allele 1. Five base changes were identified in the reverse primer binding region of the variant individual, effectively preventing annealing of the 3' primer to the template.