Frank Gannon

The impact of the PCR plateau phase on quantitative PCR

The quantitative use of the polymerase chain reaction (PCR) is often compromised by the variability of the amplification. The most useful system for quantitation by PCR involves the use of controls which are almost identical to the target and which can be amplified using the same primers as the sequences of interest. In this paper, we use a model system consisting of differently sized targets amplifiable with varying primers to demonstrate the effects of the plateau phase of PCR on quantitation by PCR. This model confirms two commonly observed results: (i) when varying amounts of a single target are amplified, a constant maximum level of product is obtained and (ii) coamplification of different concentrations of different targets results in retention of the initial proportions. The inherent contradiction in these results is examined by replacement of the key elements of the reaction including enzyme, dNTPs or primers, none of which have an effect on the plateau. Pyrophosphate is found to exert no inhibitory effect on the reaction, nor does the exonuclease action of the enzyme cause the plateau. Levels of amplification attained during amplification are both theoretically and empirically defined as being insufficient to lead to the plateau due to competition between self-annealing of product DNAs and primer binding. We conclude that, pending further biochemical enquiry into the enzyme(s) used in the PCR, none of reasons conventionally proposed for the plateau phase of the PCR are sufficient to explain the phenomenon. This being so, we define the plateau as being a feature of the reaction as a whole and, since the onset of this phase is simultaneous for all amplicons, quantitation using the internal control system need not require exponential amplification. This therefore greatly simplifies the quantitative application of PCR.

The salmon gene encoding apolipoprotein A-I: cDNA sequence, tissue expression and evolution.

A cDNA encoding an apolipoprotein (Apo) has been isolated from the Atlantic salmon (Salmo salar) and sequenced. It encodes a peptide of 258 amino acids (aa), including a signal peptide of 18 aa, with 5- and 3-untranslated regions of the mRNA of 12 and 329 nucleotides, respectively. The protein has structural features in common with other Apos of human and avian origin, including conserved sequences in the signal peptide and a series of internal repeats of 22 aa. The sequence has been identified as salmon Apo A-I (sApoA-I), and has 23% aa identity with human ApoA-I. Northern-blot analysis using the sApoA-I cDNA probe against total RNA prepared from several salmon tissues detects the expression of this gene in liver, intestine and muscle. A phylogenetic analysis reveals that the mammalian ApoA-I, ApoA-IV and Apo-E aa sequences are more closely related to each other than any of them are to sApoA-I. This suggests that the duplication events, from which A-I, A-IV and E arose, occurred after the divergence of the tetrapod and teleost ancestors.

The expression of a foreign gene in salmon embryos.

Abstract DNA which contained the E. coli β-galactosidase gene fused to the mouse metallothionein promoter was microinjected into fertilized ova from Atlantic salmon (Salmo salar). Nine to 14 weeks later, embryos which derived from the injected ova were shown to express β-galactosidase activity. The DNA which had been microinjected into the ova was also present in some embryos after 14 weeks development. It was not possible to show if the injected DNA had in fact integrated into the chromosomes. This study provides evidence for the functioning of the mouse metallothionein promoter in vivo in salmon embryos.

Use of polymerase chain reaction for early identification of Mycobacterium tuberculosis in positive cultures.

AIMS: To develop a readily applicable polymerase chain reaction (PCR) based technique which would permit the identification of Mycobacterium tuberculosis complex isolates from Bactec phials at an earlier stage than currently available methods. METHODS: Mycobacterial cells cultured in Bactec 12B medium were harvested by centrifugation. The cells were lysed by heating in distilled water. Oligonucleotide primers based on the sequence of the gene coding for the immunogenic protein MPB64 were then used to amplify a 240 base pair fragment of DNA directly from the crude cell lysate. The PCR product was visualised under ultraviolet light following electrophoresis of an aliquot in an agarose gel containing ethidium bromide. The sensitivity of the PCR was adjusted so that about 600 cfu of M tuberculosis gave a positive result. The lowest growth index at which this method of identification might be applied to Bactec phials was determined and a number of routine cultures giving a positive growth index examined. RESULTS: M tuberculosis was positively identified at the lowest growth index, as determined by the Bactec system. Of 45 routine cultures examined, with growth indexes ranging from 6 to 999, the 15 confirmed by conventional means to contain M tuberculosis were correctly identified from 1 ml of culture medium. CONCLUSIONS: The method described can be used to identify M tuberculosis isolates cultured in the Bactec system at the earliest detectable rise in growth index. It may therefore allow cultured mycobacteria to be identified at an earlier stage than conventional methods or the commercially available DNA probes adapted for use with the Bactec system.

Effects of electropermeabilization on the association of foreign DNA with pig sperm

This study was conducted to evaluate the effects of an electric pulse (electroporation/electropermeabilization) on the binding of foreign DNA molecules to porcine spermatozoa. We previously examined various parameters involved in the association of foreign DNA with sperm after a simple incubation procedure and now report the effects of electroporation on this association. Using end-labeled and random primer labeled lambda HindIII DNA fragments (23-0.125 kb), it was demonstrated that the DNA fragments interacted with the sperm after electroporation. These samples were then centrifuged and washed extensively to establish if any of the labeled DNA was associated with the spermatozoa. It was determined that approximately 10(8) molecules of DNA were associated with 1.5 x 10(7) mL-1 motile spermatozoa after five medium washes. After each wash, samples were withdrawn for gel analysis and scintillation counting. Gel analysis followed by autoradiography revealed the distinctive band pattern of lambda HindIII DNA associating with sperm. In situ visualization studies with biotin-labeled DNA revealed that approximately 75% of motile sperm carried DNA bound to the post-acrosomal region. However, the intensity of the binding varied, with some sperm being more strongly stained than others. Using [3H]dCTP-labeled DNA followed by light microscope autoradiography, approximately 70% of the sperm were strongly stained in the post-acrosomal region. There was a 5-10% increase in the amount of DNA bound by sperm when the samples were electroporated.

Tail-to-tail orientation of the Atlantic Salmon alpha- and beta-globin genes

We report the cloning of a cDNA and two corresponding β-globin genes of the Atlantic salmon (Salmo salar L.) as well as two genes for α-globins. Nucleotide sequence analysis of the cDNA shows that the predicted β-globin peptide comprises 148 amino acids with a calculated molecular mass of 16,127 Da and an overall amino acid similarity of 40–50% to higher vertebrates and 60–90% to fish sequences. The study of the genomic organization of α- and β-globin genes shows that, as is the case in Xenopus, the salmon genes are adjacent. Two sets of linked α- and β-globin genes were isolated and restriction-enzyme polymorphisms indicate that they belong to two distinct loci, possibly as a result of the salmon tetraploidy. In each locus the α- and β-globin genes are oriented 3′ to 3′ relative to each other with the RNA coding sequences located on opposite DNA strands. This is the first evidence for this type of arrangement found for globin genes. Moreover, while the linkage found in salmon and Xenopus supports the hypothesis of an initial tandem duplication of a globin ancestor gene, our results raise the question of the actual original orientation of the duplicated genes.

Sequence analysis of the 5′ flanking region of the human estrogen receptor gene

We present the sequence of 2770 nucleotides of 5 flanking sequence of the human estrogen receptor (hER) gene. The positions of potential binding sites for a number of trans-acting factors including Sp1, OTF-1, INR, TATA and CAAT box factors as well as several half palindromic hormone responsive elements (HREs) have been mapped by comparison with the consensus binding sequences. A long alternating purine/pyrimidine (APP) tract which has the potential for structural diversity as indicated by site-specific cleavage with S1 nuclease is another feature of this region. The organization of this promoter region is compared to that of other cloned members of this family. The potential roles that these sequences may play in the transcriptional regulation of this gene are discussed.

The ribosomal intergenic spacer region: a target for the PCR based diagnosis of tuberculosis.

SETTINGnDNA Diagnostics, The National Diagnostics Centre and University College Hospital, Galway, Republic of Ireland.nnnOBJECTIVEnTo investigate the possibility of developing a DNA probe to distinguish the members of the Mycobacterium tuberculosis complex and develop an assay for the detection of M. tuberculosis in sputum.nnnDESIGNnThe ribosomal intergenic spacer regions from the members of the M. tuberculosis complex were sequenced and analysed for the ability of this region to provide sequence diversity for DNA probe development.nnnRESULTSnThe 100% sequence homology in this region precluded the development of a probe to distinguish the individual members of the M. tuberculosis complex. However it was possible to develop a DNA probe that could specifically detect only the members of the M. tuberculosis complex.nnnCONCLUSIONnA specific DNA probe was developed for the detection of the M. tuberculosis complex and its application to M. tuberculosis detection in sputum was demonstrated.

Sequence analysis of a second cDNA encoding atlantic salmon (Salmo salar) serum albumin

Two similar, but distinct, cDNAs for Atlantic salmon serum albumin have been isolated from the same salmon liver. Comparison between the asSA-1 and asSA-2 sequences reveals 1% overall sequence difference.

Evaluation of a PCR assay for detection of Mycobacterium tuberculosis in clinical specimens.

A polymerase chain reaction (PCR) assay for detection of M. tuberculosis was optimized for application to clinical specimens, which were prepared for amplification by boiling in buffer. The buffer contained a synthetic DNA fragment to determine if DNA amplification from the individual prepared specimens was subject to inhibition because of substances present in the specimen, or by the process of specimen preparation or storage. The PCR test was less sensitive than direct microscopy (75% as against 87.5%) and had a specificity of 97%. Invalid results due to inhibition of amplification occurred in 12% of specimens. Incorporation of the internal standard into the specimen preparation buffer ensures that any step in the process which inhibits DNA amplification is detected in the failure of amplification of the internal standard. The use of internal standard in this way should be considered in developing diagnostic protocols.