Presence of bacterial phage-like DNA sequences in commercial Taq DNA polymerase reagents

Many studies have reported the presence of bacterial DNA contamination in commercial Taq DNA polymerase reagents. This is the first report of the presence of phage-like DNA sequences in certain commercial Taq DNA polymerase reagents. Precautions are needed when using amplification reagents with exogenous DNAs.     

Identification of related DNA sequences in Borrelia burgdorferi and two strains of Leptospira interrogans by using polymerase chain reaction.

The suitability of a polymerase chain reaction assay for Borrelia burgdorferi in epidemiological studies of infected tick populations was evaluated by using 28 strains of Leptospira interrogans and lysates of fixed adult Ixodes tick tissues. Two false positives representing leptospires were differentiated from B. burgdorferi by using an oligonucleotide probe.     

自组装短肽K_2I_4K_2在PCR中提高Taq DNA聚合酶热稳定性

目的设计自组装短肽K_2I_4K_2作为新型表面活性剂来稳定Taq DNA聚合酶,探索其对Taq DNA聚合酶在高温下的半衰期以及对PCR扩增效率的影响,探讨其在PCR中应用的可行性。方法圆二色仪检测短肽K_2I_4K_2在不同理化条件下的二级结构;原子力显微镜检测其自组装形成的纳米结构特征;紫外可见光谱检测其对Taq酶在高温下半衰期的影响;普通PCR验证其在PCR中应用的可行性;RT-q PCR检测其对Taq酶扩增效率的影响。结果短肽K_2I_4K_2在盐离子溶液中能自组装形成纳米纤维结构,且在高温下拥有稳定的二级结构;短肽K_2I_4K_2使得Taq在95℃下的半衰期增加了约1.6倍;短肽K_2I_4K_2组装24 h后大大增加了Taq酶的扩增效率。结论短肽K_2I_4K_2较为显著地增加了Taq DNA聚合酶的热稳定性,能够很好地应用于PCR反应系统中。     

Identification of mycobacteria from animals by restriction enzyme analysis and direct DNA cycle sequencing of polymerase chain reaction-amplified 16S rRNA gene sequences.

Two methods, based on analysis of the polymerase chain reaction-amplified 16S rRNA gene by restriction enzyme analysis (REA) or direct cycle sequencing, were developed for rapid identification of mycobacteria isolated from animals and were compared to traditional phenotypic typing. BACTEC 7H12 cultures of the specimens were examined for "cording," and specific polymerase chain reaction amplification was performed to identify the presence of tubercle complex mycobacteria. Combined results of separate REAs with HhaI, MspI, MboI, and ThaI differentiated 12 of 15 mycobacterial species tested. HhaI, MspI, and ThaI restriction enzyme profiles differentiated Actinobacillus species from mycobacterial species. Mycobacterium bovis could not be differentiated from M. bovis BCG or Mycobacterium tuberculosis. Similarly, Mycobacterium avium and Mycobacterium paratuberculosis could not be distinguished from each other by REA but were differentiated by cycle sequencing. Compared with traditional typing, both methods allowed rapid and more accurate identification of acid-fast organisms recovered from 21 specimens of bovine and badger origin. Two groups of isolates were not typed definitively by either molecular method. One group of four isolates may constitute a new species phylogenetically very closely related to Mycobacterium simiae. The remaining unidentified isolates (three badger and one bovine) had identical restriction enzyme profiles and shared 100% nucleotide identify over the sequenced signature region. This nucleotide sequence most closely resembled the data base sequence of Mycobacterium senegalense.     

Detection of pseudorabies virus DNA sequences by the polymerase chain reaction

Summary Genomic sequences of pseudorabies virus, a porcine herpesvirus, were amplified by the polymerase chain reaction from cells of infected cultures, nasal cells and organs from acutely diseased as well as from organs of latently infected pigs.On leave from: Department of Epizootiology, University of Veterinary Sciences, Budapest, Hungary.On leave from: Department of Agricultural Chemical Technology, Technical University, Budapest, Hungary.     

Differences in genomic DNA sequences between pathogenic and nonpathogenic isolates of Entamoeba histolytica identified by polymerase chain reaction.

A lambda gt11 cDNA library was constructed from the poly(A)+ RNA of trophozoites of Entamoeba histolytica HM-1:IMSS strain. The library was immunologically screened with monoclonal antibody 4G6, which is specific for the 30,000-Mr antigen of pathogenic isolates. A 0.7-kb clone was isolated, and its nucleotide sequence was determined. To examine whether this gene was specific for pathogenic isolates, a polymerase chain reaction was performed by using four sets of primers and the genomic DNA of pathogenic and nonpathogenic isolates as templates. Amplified DNAs were detected not only in pathogenic isolates but also in nonpathogenic isolates. However, when sequences of amplified DNA of these isolates were compared, minor differences were observed. By considering the presence or absence of recognition sites of some endonucleases, it was possible to distinguish between the pathogenic and nonpathogenic isolates. When various isolates with different zymodemes were examined by polymerase chain reaction and enzyme digestion, the results of typing were entirely in accord with those of zymodeme analysis. These results indicate that there is dimorphism in the genomic DNA coding the 30,000-Mr antigen of E. histolytica and that the combined use of the polymerase chain reaction and enzyme digestion is a useful strategy for identification of species and determination of pathogenicity.     

Detection of chicken anaemia agent DNA sequences by the polymerase chain reaction

Summary A polymerase chain reaction (PCR) assay was developed for detection of chicken anaemia agent (CAA) DNA. The assay used a single set of 20-base primers complementary to sequences located in the coding regions of the CAA replicative form (RF) DNA genome at positions 485 to 504 and 1048 to 1067. The observed amplification product had the expected size of 583 bp and was confirmed to derive from CAA RF DNA by a unique Hind III restriction enzyme cleavage pattern. The amplified fragment was shown to be specific for CAA RF DNA after chemiluminescence dot blot hybridisation with a digoxigeninlabelled 25-base internal probe. The optimised PCR assay was specific for CAA and highly sensitive, being able to detect a single CAA-infected MDCC-MSB1 cell and at least 100 fg of CAA RF DNA. Preliminary results also showed that the PCR assay can detect CAA DNA in clinical specimens from chicks experimentally infected with CAA.     

Identification of human papillomavirus DNA sequences in peripheral blood mononuclear cells

Polymerase chain reaction (PCR) was used to amplify and identify the presence of the DNA of human papillomavirus (HPV) types 6, 11, 16, and 18 in peripheral blood mononuclear cells (PBMCs) of women with and without urogenital HPV infections. HPV DNA of various types was found in PBMCs of 13 of 25 (52.0%) patients with urogenital HPV infections and in none of the 19 control subjects who are free of urogenital HPV infections. The presence of HPV DNA in PBMCs may impair the immunologic functions of the lymphocytes and play a role in the epidemiology of HPV infections and the pathogenesis of HPV-induced diseases.     

Varicella-zoster virus-induced DNA polymerase.

Nuclear extracts of varicella-zoster virus (VZV)-infected human embryo lung (HEL) cells were found to contain DNA polymerase activity not present in uninfected HEL cells. This enzyme was designated the VZV-induced DNA polymerase. The VZV-induced polymerase was partially separated from the cellular alpha- and beta-polymerases by fractionation of the cells and by phosphocellulose chromatography. The separated enzymes were examined for the effect of added (NH4)2SO4, activity with synthetic templates, optimal pH, and the effect of phosphonoacetic acid. The VZV-induced DNA polymerase was distinct from cellular enzymes and had the properties of a typical herpesvirus-induced DNA polymerase.     

Detection of human papillomavirus type 16 DNA sequences in archival cervical tissues by the polymerase chain reaction.

We have evaluated the polymerase chain reaction for the detection of viral DNA sequences in paraffin-embedded archival tissues. In 63 frozen cervical biopsy specimens that were taken from premalignant and invasive lesions, Southern blotting detected human papillomavirus (HPV) type 16 DNA in 28 (44%) of the samples. In the polymerase chain reaction analysis of the formalin-fixed, paraffin-embedded mirror biopsy specimens, 46 (73%) of the tissues were found to be positive for HPV type 16. In three Southern blotting-positive cases, the DNA of the paraffin-embedded sections was too scant or too degraded to allow the detection of HPV DNA by the polymerase chain reaction. In 21 Southern blotting-negative cases, HPV type 16 DNA could be demonstrated in the archival sections by the polymerase chain reaction technique--a sensitivity improvement of more than 80% over the standard method of HPV detection in tissues.     

Identification of autonomous replication sequences in genomic and mitochondrial DNA of Crithidia fasciculata

High frequency transformation of Saccharomyces cerevisiae was used as a functional assay to isolate autonomous replication sequences (ars) from the genomic and kinetoplast DNA of the insect trypanosomatid Crithidia fasciculata. Three independent cloned genomic sequences and one kinetoplast DNA sequence promoted high frequency transformation and extrachromosomal maintenance of the YIp5 plasmid DNA in yeast. The kinetoplast DNA clone was sub-cloned to further localize the DNA sequence essential for ars activity. This element was shown to be contained in a 2 kb HindIII-EcoRI fragment derived from a 8 kb HindIII fragment of the maxicircle component of the kinetoplast DNA. This 2 kb fragment is within a DNA sequence that has been shown to strongly hybridize to Trypanosoma brucei maxicircle DNA.     

Detection of human papillomavirus and Epstein-Barr virus DNA sequences in oral mucosa of HIV-infected patients by the polymerase chain reaction.

The presence of human papillomavirus (HPV) and Epstein-Barr virus (EBV) was analyzed in 21 oral biopsy specimens of HIV-infected patients using the polymerase chain reaction (PCR) method. Biopsies were categorized as hairy leukoplakia (HL) (n = 12), candidiasis (n = 3), oral warts (n = 2), and clinically normal epithelium (n = 4). For HPV detection a modified general primer-mediated PCR method (GP-PCR), which detects a broad spectrum of HPV genotypes at sub-picogram levels, was used. Human papillomavirus DNA was only found in two oral warts and was identified as HPV type 32. Epstein-Barr virus DNA was detected in 16 biopsy specimens, including the 12 HLs, 2 cases of candidiasis, and 2 samples of normal epithelium. Epstein-Barr virus positivity in HL could be confirmed by Southern blot analysis and DNA in situ hybridization using biotinylated DNA probes (bio-DISH). Epstein-Barr virus bio-DISH was also positive in one sample of normal epithelium from a patient with HL. The results indicate that HL is strongly associated with EBV and not with any of the common HPV types that react with general HPV primers in the PCR. However the detection of EBV in normal oral epithelium by PCR and bio-DISH suggests that the presence of this virus is not exclusively related to HL.     

Identification of Lens culinaris ssp. culinaris chromosomes by physical mapping of repetitive DNA sequences

We describe the characterisation and the chromosomal localisation of two repeated DNA sequences, named pLc30 (466 bp long, 64% AT residues) and pLc7 (408 bp long, 61% AT residues), isolated from lentil (Lens culinaris ssp. culinaris) genomic DNA. The pLc30 family is characterised by four internal repeats organised in a head-to-tail orientation, whereas the pLc7 contains many short direct subrepeats. The two families do not share significant sequence similarity. The distribution of these repetitive sequences in different Lens species and in other legumes was investigated. pLc30 is present in all Lens species investigated but absent from other genera examined. In contrast, pLc7 is present also in the genome of other legumes. As determined by FISH, the pLc30 sequence hybridises on six out of seven lentil chromosome pairs, while pLc7 hybridises on one only. The distribution of the nine different hybridisation sites of pLc30 allows the discrimination of all seven chromosome pairs and the construction of a karyotype of L. culinaris ssp. culinaris. Additionally, the combination of simultaneous and successive FISH with pLc7, 5S rRNA, 18S-5.8S-25S rRNA genes, and a telomeric sequence allowed the assembly of a physical map based on lentil karyotype.     

Elimination of Bacterial DNA from Taq DNA Polymerases by Restriction Endonuclease Digestion

The incidence of false positives due to the presence of bacterial DNA in Taq DNA polymerase is an obstacle to the use of PCR in the diagnosis of infection. We describe a method that uses a restriction enzyme to destroy the ability of contaminating sequences to act as templates for a nested PCR which uses primers based on the 16S rRNA genes. The method was used prior to a PCR that amplified 10 fg of bacterial DNA. This method can be readily adapted to suit other sensitive PCRs required for clinical applications.     

Identification of the yeast DNA polymerase I gene with antibody probes

Summary Partially overlapping fragments of the gene encoding yeast DNA polymerase I have been cloned by immunological screening of a yeast genomic library constructed in the phage expression vector gt11. The three gene fragments we analyzed in detail encode part of a yeast protein that has been identified as yeast DNA polymerase I, because it shares with this enzyme a number of antigenic determinants. In fact, the yeast protein fragments expressed by the recombinant phages react with both polyclonal and monoclonal antibodies raised against different, highly purified preparations of DNA polymerase I. Moreover, they can be used to affinity purify antibodies specifically reacting with active DNA polymerase I polypeptides and they compete with the yeast enzyme for binding to antibodies that inhibit catalytic activity. The gene is located on chromosome XIV in the yeast genome, and it is transcribed as a 5.2 kb mRNA.     

Identification of visceral Leishmania species with cloned sequences of kinetoplast DNA

Several efforts have been made in order to develop more precise and sensitive methods in the identification of Leishmania parasites. We report here the identification of cloned subfragments of minicircle kinetoplast DNA (kDNA) isolated from L. donovani, WR352, which show different taxonomic specificities. Analysis of these fragments demonstrates a significant sequence diversity within the kDNA minicircle. For example, one cloned fragment was found to be present in all visceral Leishmania species tested, but was not present in any of the cutaneous Leishmania species. Another cloned fragment was only found in the strain from which it had been derived, and was not present in any of the other strains tested. In similar experiments with the New World visceral leishmanias (L. chagasi, WR518) several different cloned kDNA fragments were found to react with all of isolates of the L. chagasi tested, but not with any cutaneous Leishmania species, either from the Old World or the New World. It is of interest to note that these cloned L. chagasi kDNA fragments reacted with isolates of African visceral Leishmania species but not with isolates from India.     

Inhibition of Taq polymerase as a method for screening heparin for oversulfated contaminants

Heparin and low molecular heparins are extensively used in the treatment of a wide range of diseases in addition to their classic anticoagulant activity and can be found coating medical devices such as catheters, stents and filters. Early in 2008, a sharp increase in heparin-associated severe adverse events, including over 80 deaths, was linked to the presence of a contaminant identified as hypersulfated chondroitin sulfate (OS-CS). OS-CS is one of several oversulfated glycosaminoglycans (GAGs) of different origins that can potentially cause similar clinical problems underscoring the need to develop robust screening methods for contaminants in existing and future lots of heparin. This study demonstrates that oversulfated GAGs block the activity of Taq polymerase used for real time PCR. Based on this finding we developed a simple, rapid, sensitive and high throughput screening method to detect and quantify oversulfated chondroitin sulfate (OS-CS) and other potential oversulfated contaminants in commercial lots of heparin. This method requires less than 100 miliUnits (mU) of heparin as starting material, therefore avoiding the need to lyophilize and concentrate samples, and has a limit of detection of <1 ng for all oversulfated GAGs tested.