Showing papers on "genomic DNA published in 1990"

PCR protocols : A guide to methods and applications

Abstract: Basic Methodology: M.A. Innis and D.H. Gelfand, Optimization of PCRs. R.K. Saiki, Amplification of Genomic DNA. E.S. Kawasaki, Amplification of RNA. M.A. Frohman, RACE: Rapid Amplification of cDNA Ends. T. Compton, Degenerate Primers for DNA Amplification. C.C. Lee and C.T. Caskey, cDNA Cloning Using Degenerate Primers. M.A. Innis, PCR with 7-Deaza-2~b7-Deoxyguanosine Triphosphate. G. Gilliland, S. Perrin, and H.F. Bunn, Competitive PCR for Quantitation of mRNA. A.M. Wang and D.F. Mark, Quantitative PCR. P.C. McCabe, Production of Single-Stranded DNA by Asymmetric PCR. S.J. Scharf, Cloning with PCR. U. Landegren, R. Kaiser, and L. Hood, Oligonucleotide Ligation Assay. C. Levenson and C.-A. Chang, Nonisotopically Labeled Probes and Primers. Y-M.D. Lo, W.Z. Mehal, and K.A. Fleming, Incorporation of Biotinylated dUTP. R. Helmuth, Nonisotopic Detection of PCR Products. D.H. Gelfand and T.J. White, Thermostable DNA Polymerases. S. Kwok, Procedure to Minimuze PCR-Product Carry-Over. E.S. Kawasaki, Sample Preparation from Blood, Cells, and Other Fluids. D.K. Wright and M.M. Manos, Sample Preparation from Paraffin-Embedded Tissues. S. P~ada~adabo, Amplifying Ancient DNA. Research Applications. M.J. Holland and M.A. Innis, In Vitro Transcription of PCR Templates. R. Higuchi, Recombinant PCR. B. Krummel, DNase I Footprinting. M.A.D. Brow, Sequencing with Taq DNA Polymerase. S.S. Sommer, G. Sarkar, D.D. Koeberl, C.D.K. Bottema, J.-M. Buerstedde, D.B. Schowalter, and J.D. Cassady, Direct Sequencing with the Aid of Phage Promoters. V.C. Sheffield, D.R. Cox, and R.M. Myers, Identifying DNA Polymorphisms by Denaturing Gradient Gel Electrophoresis. H. Ochman, M.M. Medhora, D. Garza, and D.L. Hartl, Amplification of Flanking Sequences by Inverse PCR. M.A. Frohman and G.R. Martin, Detection of Homologous Recombinants. L.M. Powell, RNA Processing: Apo-B. T.R. Gingeras, G.R. Davis, K.M. Whitfield, H.L. Chappelle, L.J. DiMichele, and D.Y. Kwoh, A Transcription-Based Amplification System. K.D. Friedman, N.L. Rosen, P.J. Newman, and R.R. Montgomery, Screening of ~glgt11 Libraries. Genetics and Evolution. H.A. Erlich and T.L. Bugawan, HLA DNA Typing. J.S. Chamberlain, R.A. Gibbs, J.E. Ranier, and C.T. Caskey, Multiplex PCR for the Diagnosis of Duchenne Muscular Dystrophy. S.B. Lee and J.W. Taylor, Isolation of DNA from Fungal Mtcelia and Single Spores. S.C. Kogan and J. Gitschier, Genetic Prediction of Hemophilia A. U. Gyllensten, Haplotype Analysis from Single Sperm or Diploid Cells. M.L. Sogin, Amplification of Ribosomal RNA Genes for Molecular Evolution Studies. T.J. White, T. Bruns, S. Lee, and J. Taylor, Amplification and Direct Sequencing of Fungal Ribosomal RNA Genes for Phylogenetics. Diagnostics and Forensics. G.D. Ehrlich, S. Greenberg, and M.A. Abbott, Detection of Human T-Cell Lymphoma/Leukemia Viruses. D.E. Kellogg and S. Kwok, Detection of Human Immunodeficiency Virus. I. Baginski, A. Ferrie, R. Watson, and D. Mack, Detection of Hepatitis B Virus. Y. Ting and M.M. Manos, Detection and Typing of Genital Human Papillomaviruses. D. Shibata, Detection of Human Cytomegalovirus. H.A. Rotbart, PCR Amplification of Enteroviruses. D. Mack, O.-S. Kwon, and F. Faloona, Novel Viruses. J. Lyons, Analysis of ras Gene Point Mutations by PCR and Olgonucleotide Hybridization. M. Crescenzi, B-Cell Lymphoma: t(14 18) Chromosome Rearrangement. R.M. Atlas and A.K. Bej, Detecting Bacterial Pathogens in Environmental Water Samples by Using PCR and Gene Probes. S.-H. Park, PCR in the Diagnosis of Retinoblastoma. C. Orrego and M.C. King, Determination of Familial Relationships. Instrumentation and Supplies: R. Watson, PCR in a Teacup A Simple and Inexpensive Method for Thermocycling PCRs. P. Denton and H. Reisner, A Low-Cost Air-Driven Cycling Oven. N.C.P. Cross, N.S. Foulkes, D. Chappel, J. McDonnell, and L. Luzzatto, Modification of a Histokinette for Use as an Automated PCR Machine. C. Orrego, Organizing a Laboratory for PCR Work. R. Madej and S. Scharf, Basic Equipment and Supplies. Index.

Rapid and simple method for purification of nucleic acids.

Abstract: We have developed a simple, rapid, and reliable protocol for the small-scale purification of DNA and RNA from, e.g., human serum and urine. The method is based on the lysing and nuclease-inactivating properties of the chaotropic agent guanidinium thiocyanate together with the nucleic acid-binding properties of silica particles or diatoms in the presence of this agent. By using size-fractionated silica particles, nucleic acids (covalently closed circular, relaxed circular, and linear double-stranded DNA; single-stranded DNA; and rRNA) could be purified from 12 different specimens in less than 1 h and were recovered in the initial reaction vessel. Purified DNA (although significantly sheared) was a good substrate for restriction endonucleases and DNA ligase and was recovered with high yields (usually over 50%) from the picogram to the microgram level. Copurified rRNA was recovered almost undegraded. Substituting size-fractionated silica particles for diatoms (the fossilized cell walls of unicellular algae) allowed for the purification of microgram amounts of genomic DNA, plasmid DNA, and rRNA from cell-rich sources, as exemplified for pathogenic gram-negative bacteria. In this paper, we show representative experiments illustrating some characteristics of the procedure which may have wide application in clinical microbiology.

Isolation of a cDNA from the virus responsible for enterically transmitted non-A, non-B hepatitis.

Abstract: Major epidemic outbreaks of viral hepatitis in underdeveloped countries result from a type of non-A, non-B hepatitis distinct from the parenterally transmitted form. The viral agent responsible for this form of epidemic, or enterically transmitted non-A, non-B hepatitis (ET-NANBH), has been serially transmitted in cynomolgus macaques (cynos) and has resulted in typical elevation in liver enzymes and the detection of characteristic virus-like particles (VLPs) in both feces and bile. Infectious bile was used for the construction of recombinant complementary DNA libraries. One clone, ET1.1, was exogenous to uninfected human and cyno genomic liver DNA, as well as to genomic DNA from infected cyno liver. ET1.1 did however, hybridize to an approximately 7.6-kilobase RNA species present only in infected cyno liver. The translated nucleic acid sequence of a portion of ET1.1 had a consensus amino acid motif consistent with an RNA-directed RNA polymerase; this enzyme is present in all positive strand RNA viruses. Furthermore, ET1.1 specifically identified similar sequences in complementary DNA prepared from infected human fecal samples collected from five geographically distinct ET-NANBH outbreaks. Therefore, ET1.1 represents a portion of the genome of the principal viral agent, to be named hepatitis E virus, which is responsible for epidemic outbreaks of ET-NANBH.

Structure and expression of the human cystatin C gene.

Abstract: The structural organization of the gene for the human cysteine-proteinase inhibitor cystatin C was studied. Restriction-endonuclease digests of human genomic DNA hybridized with human cystatin C cDNA and genomic probes produced patterns consistent with a single cystatin C gene and, also, the presence of six closely related sequences in the human genome. A 30 kb restriction map covering the genomic region of the cystatin C gene was constructed. The positions of three polymorphic restriction sites, found at examination of digests of genomic DNA from 79 subjects, were localized in the flanking regions of the gene. The gene was cloned and the nucleotide sequence of a 7.3 kb genomic segment was determined, containing the three exons of the cystatin C structural gene as well as 1.0 kb of 5'-flanking and 2.0 kb of 3'-flanking sequences. Northern-blot experiments revealed that the cystatin C gene is expressed in every human tissue examined, including kidney, liver, pancreas, intestine, stomach, antrum, lung and placenta. The highest cystatin C expression was seen in seminal vesicles. The apparently non-tissue-specific expression of this cysteine-proteinase inhibitor gene is discussed with respect to the structure of its 5'-flanking region, which shares several features with those of housekeeping genes.

Amplification of a variable number of tandem repeats (VNTR) locus (pMCT118) by the polymerase chain reaction (PCR) and its application to forensic science

Abstract: A genetic locus (D1S58, defined by DNA probe pMCT118) that contains a variable number of tandem repeats (VNTR) has been successfully amplified from a very small amount of genomic deoxyribonucleic acid (DNA) by the polymerase chain reaction (PCR). The DNA sequence of the locus was determined and was found to consist of a 16-base consensus sequence and flanking sequences. Oligonucleotide primers complementary to the flanking sequences were synthesized to serve as primers for amplification of MCT118 by the PCR method. Human genomic DNA isolated from blood (2 ng from each sample) was successfully amplified at the MCT118 locus, and polymorphic bands were detectable by ethidium bromide staining after electrophoresis on polyacrylamide gels. Determination of genotypes at this VNTR locus can now be routinely achieved within 24 h, without the need for Southern blots or radioactive materials. Furthermore, the small size (387 to 723 base pairs) of the DNA fragments produced in the PCR amplification permits good resolution of individual alleles that differ by only one repeat unit. The precise specification of the number of tandem repeats present in each allelic fragment is reproducible from one analysis to another.

Isolation of a cDNA encoding the human CD38 (T10) molecule, a cell surface glycoprotein with an unusual discontinuous pattern of expression during lymphocyte differentiation.

Abstract: A cDNA clone encoding the human lymphocyte differentiation Ag CD38 was isolated from a mixture of four different lymphocyte CDNA libraries expressed transiently in COS cells and screened by panning with mAb. Transfected COS cells expressed a surface protein of Mr 46,000 that was similar to the native CD38 molecule expressed on the B cell line Daudi and the T cell leukemia HPB-ALL and which was recognized by each of the CD38 specific mAb HIT-2, T16, T168, HB7, 5D2, ICO-18, and ICO-20. The CD38 cDNA sequence predicts an unusual 30-kDa polypeptide with a short N-terminal cytoplasmic tail, and a carboxyl-terminal extracellular domain carrying the four potential N-linked glycosylation sites. The absence of significant homology with other known surface Ag including members of the Ig superfamily ruled out the possibility that CD38 was the human homologue of the murine Qa2 molecule as has been suggested previously. PvuII digests of human genomic DNA revealed a polymorphism linked to the CD38 gene.

Characterization of four novel ras-like genes expressed in a human teratocarcinoma cell line.

Abstract: A mixed-oligonucleotide probe was used to identify four ras-like coding sequences in a human teratocarcinoma cDNA library. Two of these sequences resembled the rho genes, one was closely related to H-, K-, and N-ras, and one shared only the four sequence domains that define the ras gene superfamily. Homologs of the four genes were found in genomic DNA from a variety of mammals and from chicken. The genes were transcriptionally active in a range of human cell types.

Isolation and structural characterization of a cDNA clone encoding the human DNA repair protein for O6-alkylguanine

Abstract: O6-Methylguanine-DNA methyltransferase (MGMT; DNA-O6-methylguanine:protein-L-cysteine S-methyltransferase, EC 21163), a unique DNA repair protein present in most organisms, removes the carcinogenic and mutagenic adduct O6-alkylguanine from DNA by stoichiometrically accepting the alkyl group on a cysteine residue in a suicide reaction The mammalian protein is highly regulated in both somatic and germ-line cells In addition, the toxicity of certain alkylating drugs in tumor and normal cells is inversely related to the levels of this protein The cDNA of the human gene, henceforth named MGMT, has been cloned in an expression vector on the basis of its rescue of a methyltransferase-deficient (ada-) Escherichia coli host A 22-kDa active methyltransferase encoded entirely by the cDNA contains an amino acid sequence of 61 residues that bears 60-65% similarity with segments of E coli methyltransferase (products of the ada and ogt genes), which encompass the alkyl-acceptor residues The human cDNA has no sequence similarity with the ada and ogt genes, due in part to differences in codon usage, and shows no detectable homology with E coli genomic DNA However, it hybridizes with distinct restriction fragments of human, mouse, and rat DNAs The lack of methyltransferase observed in many human cell lines is due to the absence of the MGMT gene or to lack of synthesis and/or stability of its 095-kilobase poly(A)+ RNA transcript

The polydeoxyadenylate tract of Alu repetitive elements is polymorphic in the human genome.

Abstract: To identify DNA polymorphisms that are abundant in the human genome and are detectable by polymerase chain reaction amplification of genomic DNA, we tested the hypothesis that the polydeoxyadenylate tract of the Alu family of repetitive elements is polymorphic among human chromosomes. We analyzed the 3' ends of three specific Alu sequences and found that two (in the adenosine deaminase gene and the beta-globin pseudogene) were polymorphic. This novel class of polymorphisms, termed AluVpA [Alu variable poly(A)] may represent one of the most useful and informative group of DNA markers in the human genome.

Organization, structure, and polymorphisms of the human profilaggrin gene.

Abstract: Profilaggrin is a major protein component of the keratohyalin granules of mammalian epidermis. It is initially expressed as a large polyprotein precursor and is subsequently proteolytically processed into individual functional filaggrin molecules. We have isolated genomic DNA and cDNA clones encoding the 5'- and 3'-ends of the human gene and mRNA. The data reveal the presence of likely "CAT" and "TATA" sequences, an intron in the 5'-untranslated region, and several potential regulatory sequences. While all repeats are of the same length (972 bp, 324 amino acids), sequences display considerable variation (10-15%) between repeats on the same clone and between different clones. Most variations are attributable to single-base changes, but many also involve changes in charge. Thus, human filaggrin consists of a heterogeneous population of molecules of different sizes, charges, and sequences. However, amino acid sequences encoding the amino and carboxyl termini are more conserved, as are the 5' and 3' DNA sequences flanking the coding portions of the gene. The presence of unique restriction enzyme sites in these conserved flanking sequences has enabled calculations on the size of the full-length gene and the numbers of repeats in it: depending on the source of genomic DNA, the gene contains 10, 11, or 12 filaggrin repeats that segregate in kindred families by normal Mendelian genetic mechanisms. This means that the human profilaggrin gene system is also polymorphic with respect to size due to simple allelic differences between different individuals. The amino- and carboxyl-terminal sequences of profilaggrin contain partial or truncated repeats with unusual un-filaggrin-like sequences on the termini.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of the URA5 gene from Cryptococcus neoformans var. neoformans and its use as a selective marker for transformation.

Abstract: A cDNA encoding Cryptococcus neoformans orotidine monophosphate pyrophosphorylase (OMPPase) has been isolated by complementation of the cognate Escherichia coli pyrE mutant. The cDNA was used as a probe to isolate a genomic DNA fragment encoding the OMPPase gene (URA5). By using electroporation for the introduction of plasmid DNA containing the URA5 gene, C. neoformans ura5 mutants could be transformed at low efficiency. Ura+ transformants obtained with supercoiled plasmids containing the URA5 gene showed marked mitotic instability and contained extrachromosomal URA5 sequences, suggesting limited ability to replicate within C. neoformans. Transformants obtained with linear DNA were of two classes: stable transformants with integrated URA5 sequences, and unstable transformants with extrachromosomal URA5 sequences.

Discrimination between closely related Triticeae species using genomic DNA as a probe.

Abstract: Labelled total genomic DNA was used as a probe in combination with blocking DNA to discriminate between taxonomically closely related species in the genera Hordeum and Secale. Discrimination was possible both by Southern hybridization to size-fractionated restriction enzyme digests of genomic DNA and by in situ hybridization to chromosome preparations. To distinguish between two species (e.g. H. vulgare and H. bulbosum), genomic DNA from one species was used as the labelled probe, while unlabelled DNA from the other species was applied at a much higher concentration as a block. The blocking DNA presumably hybridized to sequences in common between the block and the labelled probe, and between the block and DNA sequences on the membrane or chromosomes in situ. If so, mainly species-specific sequences would remain as sites for probe hybridization. These species-specific sequences are dispersed and represent a substantial proportion of the genome (unlike many cloned, species-specific sequences). Consequently, rapid nonradioactive methods detected probe hybridization sites satisfactorily. The method was able to confirm the parentage of hybrid plants. It has potentially wide application in plant breeding for the detection of alien DNA transfer, and it can be easily adapted to many species.

GLI3 encodes a 190-kilodalton protein with multiple regions of GLI similarity.

Abstract: The GLI oncogene, discovered by virtue of its amplification in human tumors, encodes a sequence-specific DNA-binding protein containing five zinc fingers. We have now characterized one member of a family of GLI-related zinc finger genes. A previously identified fragment of GLI3 genomic DNA was used to localize GLI3 to chromosome 7p13 and to isolate cDNA clones. Sequence analysis of these clones and identification of the GLI3 protein by using polyclonal antisera demonstrated that GLI3 encodes a protein of 1,596 amino acids and an apparent molecular mass of 190 kilodaltons. Amino acid sequence comparison with GLI demonstrated seven regions of similarity (53 to 88% identity), with the zinc fingers representing the most similar region. Furthermore, when produced in vitro, the GLI3 protein bound specifically to genomic DNA fragments containing GLI-binding sites. Amino acid sequence comparison with the product of another member of the GLI family, the Drosophila segment polarity gene cubitus interruptus Dominant, revealed additional similarity that was not shared with GLI. These studies suggest that the GLI-related genes encode a family of DNA-binding proteins with related target sequence specificities. In addition, sequence similarity aside from the zinc finger region suggests that other aspects of function are shared among the members of this gene family.

Exon trapping: a genetic screen to identify candidate transcribed sequences in cloned mammalian genomic DNA.

Abstract: Identification and recovery of transcribed sequences from cloned mammalian genomic DNA remains an important problem in isolating genes on the basis of their chromosomal location. We have developed a strategy that facilitates the recovery of exons from random pieces of cloned genomic DNA. The basis of this "exon trapping" strategy is that, during a retroviral life cycle, genomic sequences of nonviral origin are correctly spliced and may be recovered as a cDNA copy of the introduced segment. By using this genetic assay for cis-acting sequences required for RNA splicing, we have screened approximately 20 kilobase pairs of cloned genomic DNA and have recovered all four predicted exons.

Analysis of DNA in fresh and fixed tissue by the polymerase chain reaction.

Abstract: The polymerase chain reaction (PCR) was used to amplify viral or oncogene sequences from frozen or formalin-fixed, paraffin-embedded tissue sections. Methods for preparing fixed, embedded colonic tissue for PCR amplification of c-K-ras sequences from genomic DNA and for amplification of viral DNA from other tissues, including brain, lung, and liver, were evaluated. The effect of formalin fixation on the efficiency of amplification was also determined. While there seemed to be only a modest variation in the efficiency of the PCR for amplification of single-copy human genes, regardless of the methods used for tissue preparation, amplification of viral DNA sequences against a human genomic DNA background was more efficient when the DNA was purified to some degree before amplification of the tissue. We used the PCR to examine frozen and fixed embedded tissue sections for the presence of Epstein-Barr virus (EBV) and cytomegalovirus (CMV) DNA. One patient with a heart-lung transplant succumbed to a lymphoproliferative disorder, and EBV genome was present in tissues with abnormal lymphoid infiltrates. CMV was also present in bronchial lavages from the same patient, where cytologic diagnosis was not apparent. Another patient with a liver transplant showed CMV genome in multiple liver biopsies, with negative histologic results for CMV. In vitro DNA amplification with the PCR demonstrated sensitivity superior to that of histology in detecting CMV and EBV in the cases examined.

Process for determination of a complete or a partial contents of very short sequences in the samples of nucleic acids connected to the discrete particles of microscopic size by hybridization with oligonucleotide probes

Abstract: Determination of the formula of genomic DNA, ie genome sequencing, by a hybridization with oligonucleotide probes (YU Patent Application 570/87) envisages the use of 100000 oligonucleotide probes and the same number of hybridizati­ons with 6000000 of addressed sample-clones on filters in order to determine contents of oligonucleotide sequences in each clone The process presents improvements in preparati­on of samples for hybridization and improvements which enable one to follow gene expression by determining partial or complete fragment sequences of genomic DNA, mRNA or cDNA By binding fragments of genomic DNA to discrete particles (DP) of a microscopic size which are recognizable in a step of reading experimental image, the necessity for addressed samples on filters is dispensed with and this drastically reduces automatical-robotical component of the process and allows miniaturization of the entire method from a level of industrial installation to the level of laboratory instrument Processes for binding DNA fragments to DPs recognizable in common reactions, allow elimination of cloning, ie DNA amplification in the host cells and, in a process of library forming, need for formation of 6000000 addressed samples in any one of the phases of a process for sequencing by hybridization