DNA Amplification Techniques Research Articles

HLA class II frequencies in celiac disease patients in the West of Ireland

Restriction fragment length polymorphism analysis, using a single restriction enzyme TaqI-multiple-probe system for HLA-DRB1-DQB1 and -DQA1, was used to determine HLA-DR and -DQ frequencies in 56 unrelated celiac patients and 47 unrelated controls from the west of Ireland. In addition, HLA-DPB1 allelic frequencies were determined in the same grouo of patients and controls by using the technique of enzymatic DNA amplification of the polymorphic second exon of HLA-DPB1 genes in conjunction with sequence-specific oligonucleotide probing. The results suggest that HLA-DQ rather than HLA-DR is more important in conferring susceptibility to celiac disease. Furthermore, no association between HLA-DP and celiac disease was found in this study. Human Immunology 34, 47–52 (1992)

Immunohistological detection of human parvovirus B19 in formalin-fixed, paraffin-embedded tissues.

Human parvovirus B19 is a cause of aplastic crises in patients with haemolytic anaemias, prolonged bone marrow failure in the immunosuppressed, and fetal death secondary to non-immune hydrops. The immunohistological detection of parvovirus B19 in formalin-fixed, paraffin-embedded tissues has not previously been reported, and definitive diagnosis of infection in such specimens has relied on the use of specialized DNA hybridization and amplification techniques. A new monoclonal antibody to B19 capsid proteins, R92F6, was found to be capable of labelling infected cells in paraffin-embedded tissues from all 19 cases of parvovirus-related fetal hydrops tested, and in bone marrow from a child with congenital immunodeficiency and chronic parvovirus infection. Viral antigen was detected both in cytoplasmic and in nuclear distributions using the alkaline phosphatase anti-alkaline phosphatase (APAAP) technique without preceding proteolytic digestion. The viral epitope recognized appears to be highly conserved, as specimens were obtained over a 13-year period from widely spaced locations in the U.K. Antibody R92F6 should facilitate rapid diagnosis of parvovirus B19 infection in routinely processed and archival specimens.

Restriction site generating-polymerase chain reaction (RG-PCR) for the probeless detection of hidden genetic variation: application to the study of some common cystic fibrosis mutations.

In this report we describe the use of a DNA amplification technique in which modified primers introduce a base substitution adjacent to the codon of interest and create an artificial restriction site for the detection of mutations which do not produce or modify a naturally occurring restriction site (restriction site generating-polymerase chain reaction, RG-PCR). RG-PCR was developed and applied to the screening in an Italian population sample of several relatively common cystic fibrosis mutations which are not amenable to analysis with a known restriction endonuclease: G542X, 2869insG, Y913C, N1303K, and 1717-1GA. This method, which allows the identification of virtually any single base change by restriction enzyme analysis and without the need for molecular probes, is rapid and easy to perform. The combined use of RG-PCR for several different CF mutations in multiplex tests further expands the advantages of this approach.

Embryo Sexing: A Review of Current Techniques and Their Potential for Commercial Amdication in Livestock Production

The development of embryo transfer procedures has created the potential for embryo sexing as a method of controlling the sex of offspring. Five techniques have been developed for this purpose: cytogenetic analysis, assay of X-linked enzymes, analysis of differential development rates, detection of male-specific antigens, and the use of Y-specific DNA probes. These methods are reviewed and their potential for commercial application in domestic animal production is discussed. The use of Y-specific DNA probes, particularly those that employ DNA amplification techniques, has resulted in the greatest potential for commercial application. An assay based on a bovine Y-chromosome-specific DNA sequence is described. This assay has a determination rate greater than 90% with samples as small as two cells and accurately determines sex 94% of the time. For economic and technical reasons, embryo sexing has not been widely used in conventional embryo transfer, but these procedures, particularly Y-chromosome-specific DNA amplification techniques, are quite applicable to embryo cloning and will be vital when cloning becomes commercially viable.

Prenatal Transmission of Hepatitis B Virus to Neonates Born to Serum Hepatitis B Virus DNA-Positive Mothers

Hepatitis B virus (HBV) DNA was detected by in vitro enzymatic DNA amplification techniques in 66.7% (six of nine) of hepatitis B virus surface antigen (HBsAg)-positive and in 21.1% (7 of 33) of HBsAg-negative pregnant women. Five of the HBV DNA and HBsAg-positive women and one HBV DNA-positive but HBsAg-negative woman gave birth to infants positive for serum HBV DNA at time of birth. These results suggest that HBsAg-negative pregnant women are potentially capable of transmitting HBV DNA to their infants.

Human papillomaviruses and small cell carcinoma of the uterine cervix

The in vitro DNA amplification technique of polymerase chain reaction was used to evaluate the possible presence of human papillomavirus (HPV) in small cell carcinoma of the uterine cervix. None of the 12 cases examined contain detectable amounts of either HPV type 16, 18, 31, or 33 DNA. On the other hand, HPV types 16 and 18 DNA were found in 14 (93.3%) and 9 (60.0%) of 25 invasive cervical squamous carcinoma tissues. The results seem to suggest that these types of HPV are not present or are present in extremely small quantities in cervical small cell carcinoma. Such an absence of HPV DNA makes it unlikely that these types of HPV play any etiological role in the pathogenesis of cervical small cell carcinoma.

Amplification by the polymerase chain reaction of a specific target sequence in the gene coding for Escherichia coli verotoxin (VTe variant)

Synthetic oligonucleotide primers were used in a polymerase chain reaction (PCR) protocol to target a specific sequence in the gene coding for the A subunit of Escherichia coli verotoxin (VTe-variant, VTev). This PCR protocol permits the VTe-variant target sequence to be distinguished from closely related sequences in the same coding regions for type 1, type 2, and type 2 variant E. coli verotoxins. This procedure will be a valuable adjunct to other DNA amplification techniques currently being used for molecular epidemiological studies of verotoxigenic E. coli.

Amplification by the polymerase chain reaction of a specific target sequence in the gene coding forEscherichia coliverotoxin (VTe variant)

Synthetic oligonucleotide primers were used in a polymerase chain reaction (PCR) protocol to target a specific sequence in the gene coding for the A subunit of Escherichia coli verotoxin (VTe-variant, VTev). This PCR protocol permits the VTe-variant target sequence to be distinguished from closely related sequences in the same coding regions for type 1, type 2, and type 2 variant E. coli verotoxins. This procedure will be a valuable adjunct to other DNA amplification techniques currently being used for molecular epidemiological studies of verotoxigenic E. coli.

Leishmaniasis in a domestic goat in Kenya

The pathological and electron-microscopic features of the first case of autochthonous leishmaniasis affecting a domestic goat in Kenya are described. They are similar to what have been described in man and other animals. Using a short amino-acid sequence common to all the species of leishmania as primers for kDNA synthesis, the intervening sequence of 120 bases was amplified in the goat's tissues by polymerase chain reaction (PCR). The leishmania kinetoplast DNA sequence was detected in all the different infected tissues of the goat examined. The sensitivity and specificity of this assay are discussed. The result of the assay used was consistent with the parasite being either L. major or L. aethiopica as the infecting agent. The isoenzyme studies were consistent with L. aethiopica as the strain responsible for this goat's infection. The control of leishmaniasis and its vector must take into account the potential role of animal reservoirs in the environment. Even though Kenya and other East African countries are endemic for kala-azar, the presence of kala-azar in goats is of considerable veterinary public health importance in Africa. Efforts must not be spared to identify and detect other possible animal reservoirs in the subregion. Using DNA amplification techniques, which are sensitive and specific, such as the one described in this paper, sera and other biological fluids and tissues from different animal species should be utilized for detecting additional reservoirs for leishmania parasites particularly in known endemic areas of the world.

The ligase chain reaction in a PCR world.

S i n c e its discovery in 1985, the polymerase chain reaction (PCR) has had a profound impact on detecting genetic and infectious diseases, identifying new genes, and unraveling the mysteries of protein-ligand recognition.(1-s) Its universal utility is due to the exquisite specificity of amplification and ease of cycling made possible by the cloning and careful characterization of a thermostable polymerase from Thermus aquaticus. (6,7) Likewise, cloning of a thermostable ligase enabled a new amplification method, termed ligase chain reaction (LCR), to both amplify DNA and discriminate a single base mutation. (8-1~ Although these DNA amplification techniques are new, they bring to fruition the enzymes as reagents philosophy expounded by A. Kornberg and I.R. Lehman a quarter of a century ago. Allele-specific LCR employs four oligonucleotides, two adjacent oligonucleotides which uniquely hybridize to one strand of target DNA and a complementary set of adjacent oligonucleotides, which hybridize to the opposite strand (see Fig. 1, top left). Thermostable DNA ligase will covalently link each set, provided that there is complete complementarity at the junc~ tion. (8) Because the oligonucleotide products from one round may serve as substrates during the next round, the signal is amplified exponentially, analogous to PCR amplification. A single-base mismatch at the oligonucleotide junction will not be amplified and is therefore distinguished (Fig. 1, top right). A second set of mutantspecific oligonucleotides is used in a separate reaction to detect the mutant allele. This review will give a brief introduction to (1) determinants of specificity in amplification reactions; (2) differences between thermostable and thermophilic enzymes; (3) detection of single-base substitutions in DNA diagnostics; (4) characterization of DNA ligases; (5) cloning of DNA ligases; (6) use of ligases in DNA detection; (7) other methods that use ligase, such as ligase-mediated PCR and the branch capture reaction; and (8) potential new uses of ligase in PLCR and nested LCR amplification reactions. It will close with speculations on future prospects.

Identification of a new haemophilia BM case produced by a mutation located at the carboxy terminal cleavage site of activation peptide.

We describe a novel point mutation due to C----G transversion at nucleotide 20518 in the exon VI of factor IX gene, resulting in the substitution of glycine (GGG) for arginine (CGG) at position 180 in the polypeptide. This point mutation was found in a patient with a haemophilia BM variant. We designated the altered factor IX produced by this new mutation as factor IXMadrid. This mutation blocks the cleavage site involved in the release of the activation peptide at Arg180-Val181. It also abolishes the Aval site (CTCGGG) in exon VI, which can be directly detected with the enzymatic DNA amplification technique (PCR) and offers the possibility of direct analysis in carrier and prenatal diagnosis in kindreds with this mutation.

Characterization of high-affinity ryanodine-binding sites of rat liver endoplasmic reticulum. Differences between liver and skeletal muscle

In this study, the binding of [3H]ryanodine to liver microsomal subfractions was investigated. The specific binding of [3H]ryanodine, as determined both by vacuum filtration and by ultracentrifugation, is to a single class of high-affinity binding sites with a Kd of 10 +/- 2.5 nM and density of 500 +/- 100 and 1200 +/- 200 fmol/mg of protein by the filtration and centrifugation methods respectively. [3H]Ryanodine binding reached equilibrium in about 1 min and 2 min at 36 degrees C and 24 degrees C respectively, and the half-time of dissociation at 37 degrees C was approx. 15 s. The binding of [3H]ryanodine is Ca(2+)-independent: it is slightly stimulated by NaCl, Mg2+, ATP and InsP3 but strongly inhibited by caffeine, diltiazem and sodium dantrolene. Thus the binding of ryanodine to endoplasmic reticulum membranes shares some of the characteristics of its binding to the sarcoplasmic reticulum but also differs from it in several important properties, such as its Ca(2+)-independence, its rapid association and dissociation, and its inhibition by caffeine. The structural similarities between the skeletal muscle and liver binding sites were further explored by employing in vitro DNA amplification techniques, using the known sequence of the skeletal muscle receptor as reference point. The data obtained with this method indicate that the liver does not process mRNA for the skeletal muscle ryanodine receptor.

Frequent Identification of HIV-1 DNA in Bronchoalveolar Lavage Cells Obtained from Individuals with the Acquired Immunodeficiency Syndrome

Tissue macrophages are recognized as a cellular target for infection with the human immunodeficiency virus type 1 (HIV-1). To characterize the nature of this cell-retrovirus interaction within the lower respiratory tract we analyzed fluid and cells obtained by bronchoalveolar lavage (BAL) of eight individuals with acquired immunodeficiency syndrome (AIDS) who were undergoing diagnostic fiberoptic bronchoscopy. Of these eight individuals, seven had active infection with Pneumocystis carinii; one had suspected cytomegalovirus pneumonitis. At the time of study two were receiving the antiretroviral drug zidovudine (azidothymidine [AZT]). HIV-1 could not be isolated from any of the eight samples of BAL fluid concentrated by ultracentrifugation through 20% sucrose. HIV-1 antigen (p24) was detected in one of eight samples of concentrated BAL fluid but could not be found in eight samples of media conditioned by overnight incubation with adherent BAL cells. Despite the infrequent detection of HIV-1 antigen it was possible to identify HIV-1 genomic sequences by the use of a DNA amplification technique, the polymerase chain reaction, in all eight BAL cell preparations. In BAL cells adherent for up to 5 days in culture this method detected retroviral DNA that hybridized to a complementary pair of primers located in the env and gag gene regions of HIV-1. These studies demonstrate the uniform presence of HIV-1 harboring cells within the airways of the lung in individuals with AIDS and active respiratory infection and may have implications for local organ defense.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of clinically normal appearing skin for systemic infectious diseases in human immunodeficiency virus seropositive patients

The observation of a human immunodeficiency virus (HIV)-infected patient with cutaneous ulcers which contained both fungal and viral pathogens, and in which the fungal organisms were grown in culture yet could not be demonstrated microscopically, suggested the possibilities: 1) that the skin lesions might have been primarily caused by the viral pathogen, and 2) that the concurrent presence of that fungal pathogen was coincidental and perhaps secondary to fungemia. Assuming that these postulates are valid, it is reasonable to hypothesize that fungal organisms would have been detected if perilesional or distal, clinically normal appearing, skin had been evaluated. This hypothesis could be further assessed by performing a prospective study of clinically normal appearing skin for pathogens of suspected systemic infectious diseases in HIV seropositive patients. Since the quantity of infectious pathogens in nonlesional skin may be limited, it might be difficult to grow the organisms in culture or demonstrate them microscopically using standard methods. Therefore, in addition to routine cultures and histologic evaluation, immunohistochemical techniques using monoclonal antibodies to pathogen antigens and DNA amplification techniques based upon the polymerase chain reaction should be used to enhance the detection of the infectious pathogens.

Identification of foodborne pathogens by nucleic acid hybridization

Nucleic acid hybridization methods have been developed and used to identify microorganisms in foods. Tests performed on mixed cultures save the time required to established pure cultures. Enterotoxigenic or invasive strains of foodborne bacterial pathogens are detected with probes that identify genes responsible for virulence. Hybridization tests signal the presence or absence of a particular strain or an entire genus and are especially well suited for screening foods for specific pathogens. With the colony hybridization assay format, foodborne bacteria harboring a specific gene can be enumerated. However, hybridization tests require the presence of 10 5 to 10 6 cells to yield a positive results, thereby limiting sensitivity and necessitating a time-consuming growth step. In vitro DNA amplification techniques increase the amount of DNA segments 10 5 − 10 6-fold in 2 to 3 h, thus enhancing test sensitivity.

4994370 DNA amplification technique: Jonatha Silver, Vijaya Keerikatte assigned to The United States of America as represented by the Department of Health and Human Services

4994370 DNA amplification technique: Jonatha Silver, Vijaya Keerikatte assigned to The United States of America as represented by the Department of Health and Human Services

Detection and typing of maize streak virus and other distantly related geminiviruses of grasses by polymerase chain reaction amplification of a conserved viral sequence

The application of the polymerase chain reaction DNA amplification technique to the detection and typing of isolates of maize streak virus (MSV) and other related geminiviruses of grasses is described. The oligonucleotide primers used for amplification were 17-mers which contained a number of degeneracies. An approximately 250 base pair fragment was amplified from all geminivirus-infected grass and cereal samples tested. The amplification reaction was specific, working down to a concentration of 50 fg/ml of MSV-specific plasmid-cloned DNA and with a 10(-9) dilution of MSV-infected maize DNA extract. DNA could also be amplified from distantly related geminiviruses, including two different sugarcane viruses, digitaria streak virus and another as yet uncharacterized virus of a Panicum sp. Amplified DNA from a Mauritian sugarcane isolate (SSV-M) was cloned and sequenced. Sequence comparison and phylogenetic analysis showed that this sequence differed sufficiently from the analogous region of other geminiviruses for SSV-M to be considered a distinct virus. The use of the polymerase chain reaction for the amplification of gemini- and other virus genomes or genomic fragments for typing, mapping, phylogenetic analysis and taxonomy is discussed.

A rhesus monkey model for sexual transmission of a papillomavirus isolated from a squamous cell carcinoma.

Recently we molecularly cloned and characterized a papillomavirus from a lymph node metastasis of a primary penile carcinoma found in a rhesus monkey; this virus species, rhesus papillomavirus type 1 (RhPV-1), is similar to oncogenic human papillomaviruses (HPVs), such as HPV-16 or HPV-18, in that the RhPV-1 DNA was found to be integrated in the tumor cell DNA. To compare the sexual transmission and oncogenic nature of RhPV-1 with these HPVs, we undertook an extensive retrospective study of a group of rhesus monkeys whose sexual mating and offspring histories were known. These animals had mated directly with the index male mentioned above or were secondarily exposed to this virus through intermediate sexual partners. This study combines cytological, histopathological, and several complementary hybridization and DNA amplification techniques on multiple tissue samples to demonstrate the sexually transmitted nature of RhPV-1. The oncogenic potential of RhPV-1 is suggested in several of the infected animals by the presence of various degrees of neoplasia including squamous cell cancer of the cervix.

A point mutation in the tyrosinase gene of BALB/c albino mouse causing the cysteine → serine substitution at position 85

Murine albinism is characterized by complete lack of melanin pigments in skin and retina. In order to study the molecular basis of albinism, we have cloned and characterized the tyrosinase gene of BALB/c mice (c/c). Sequence analysis of this gene reveals a point mutation at nucleotide residue 387 (G----C transversion) causing a Cys----Ser substitution at position 85 in one of the cysteine-rich domains of the tyrosinase molecule. Since this G----C transversion creates an additional DdeI site, we were able to confirm that this mutation is actually present in BALB/c genomic DNA using DNA amplification techniques. In contrast, both C57BL/6 (C/C) and DBA/2 (C/C) mouse strains carry the G residue at the same position, suggesting that this point mutation is specific for the albino mutation at the c locus. Moreover, we were able to show that the tyrosinase containing Ser-85 is not functional in transient expression of its cDNA. We therefore suggest that a G----C transversion at nucleotide residue 387 of the tyrosinase gene could lead to the albino phenotype of BALB/c mouse.

New Rapid Detection Methods for Salmonella and Listeria

We are now very much aware of the hazards associated with salmonella and listeria in foodstuffs. Newer methodologies are compared with conventional techniques for their rapid detection. Recent developments in culture media are discussed, as is a new rapid culture test kit for salmonella. Novel techniques such as ELISA, nucleic acid probes and flow cytometry are described, and a comparative assessment is made of reported findings. Nucleic acid probe and ELISA assays are considerably quicker than traditional culture techniques but they would benefit from increased sensitivity; they show inferior specificity with listeria and are still dependent on an enrichment stage. Recent DNA amplification techniques could provide a solution to the problem of poor probe sensitivity and allow the direct detection of pathogens from food samples. Flow cytometry may have the potential for high sensitivity and specificity and could be useful for on‐line monitoring in factories.