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N Arnheim

Publications and source records attributed to N Arnheim.

At least 91 records · Page 5Linked to original sources
PubMed1989

A simple method for site-directed mutagenesis using the polymerase chain reaction.

We have developed a general and simple method for directing specific sequence changes in a plasmid using primed amplification by the polymerase chain reaction (PCR). The method is based on the amplification of the entire plasmid using primers that include the desired changes. The method is rapid, simple in its execution, and requires only minute amounts of plasmid template DNA. It is significant that there are no special requirements for appropriately placed restriction sites in the sequence to be manipulated. In our system the yield of transformants was high and the fraction of them harboring plasmids with only the desired change was consistently about 80%. The generality of the method should make it useful for the direct alteration of most cloned genes. The only limitation may be the total length of the plasmid to be manipulated. During the study we found that the Taq DNA polymerase used for PCR adds on a single extra base (usually an A) at the end of a large fraction of the newly synthesized chains. These had to be removed by the Klenow fragment of DNA polymerase to insure restoration of the gene sequence.

Base Sequence↗
PubMed1989

Detection of human papillomavirus DNA in fine-needle aspirations of metastatic squamous-cell carcinoma of the uterine cervix using the polymerase chain reaction.

Routinely processed fine-needle aspirations of metastatic squamous-cell carcinoma (SCC) were analyzed for human papillomavirus type 16 (HPV-16) using the polymerase chain reaction (PCR), an in vitro DNA amplification method. HPV-16 DNA was detected in five of seven (71%) metastases from SCC of the uterine cervix. In two cases in which the primary tumor was available for comparison, the HPV-16 DNA content of the primary tumor and of the metastasis was identical. HPV-16 was not found in a metastatic SCC from the lung or in a metastatic nasopharyngeal SCC. These findings demonstrate that HPV-16 DNA sequences can be readily detected in routinely processed fine-needle aspirations using the polymerase chain reaction. The finding of HPV-16 DNA in a metastasis may serve to direct a search for a primary site of origin.

Adolescent↗
PubMed1989

The polymerase chain reaction.

The polymerase chain reaction (PCR) is a powerful new method for 'in vitro cloning'. It can selectively amplify a single molecule of template DNA several millionfold in a few hours and has made possible new approaches to problems in molecular genetics, evolutionary biology, and development.

Biological Evolution↗
PubMed1989

Single-sperm typing: determination of genetic distance between the G gamma-globin and parathyroid hormone loci by using the polymerase chain reaction and allele-specific oligomers.

The frequency of recombination between the G gamma-globin (HBG2) and parathyroid hormone (PTH) loci on the short arm of human chromosome 11 was estimated by typing greater than 700 single-sperm samples from two males. The sperm-typing technique employed involves the polymerase chain reaction and allele-specific oligonucleotide hybridization. Our maximum likelihood recombination fraction estimate of 0.16 (95%) confidence interval, 0.13-0.19) falls well within previous estimates based on family studies. With current technology and a sample size of 1000 sperm, recombination fractions down to approximately 0.009 can be estimated with statistical reliability; with a sample size of 5000 sperm, this value drops to about 0.004. Reasonable technological improvements could result in the detection of recombination frequencies less than 0.001.

Alleles↗
PubMed1989

Detection of human papillomavirus in formalin-fixed, invasive squamous carcinomas using the polymerase chain reaction.

We analyzed 88 formalin-fixed, paraffin-embedded invasive squamous carcinomas for human papillomavirus-related DNA sequences (HPV types 16 and 18) following in vitro gene amplification using the polymerase chain reaction. HPV DNA sequences were found in 35 of 50 (70%) carcinomas of the anogenital region, including four of four (100%) anal, six of eight (75%) vulvar, nine of 14 (64%) vaginal, two of five (40%) penile, and 14 of 19 (74%) cervical tumors. Nine of 25 (36%) oropharyngeal squamous carcinomas contained HPV DNA sequences, including four of 10 (40%) laryngeal, three of eight (38%) buccal, and two of seven (29%) glossal tumors. HPV DNA sequences were not found in 13 esophageal carcinomas. Of the 44 cases that contained viral DNA, HPV-16 was detected in 41 cases (93%) and HPV-18 in five cases (11%), while both types were found in two cases (one anal and one vulvar). HPV DNA sequences were found in 43 of 83 (52%) nonverrucous and in one of five (20%) verrucous carcinomas, but this difference was not significant. These findings demonstrate that HPV DNA sequences are more frequently associated with anogenital than oropharyngeal squamous carcinomas and can be readily detected in routinely processed tissues using the polymerase chain reaction.

Blotting, Southern↗
PubMed1989

Fine-structure genetic mapping of human chromosomes using the polymerase chain reaction on single sperm: experimental design considerations.

The polymerase chain reaction (PCR) makes it possible to rapidly generate a very large number of copies of a specific region of DNA. Application of PCR to individual human sperm cells permits the typing of a large number of independent male meiotic events. If the donor male is heterozygous at three loci, sperm typing using PCR will permit ordering of loci in a manner analogous to classical methods of experimental genetics. Sequential analysis of trios of loci by sperm typing will provide a very accurate means of ordering any number of tightly linked loci. Here, we describe experimental design and sample-size issues raised by the application of sperm typing by PCR for mapping human chromosomes, and we demonstrate that sperm typing will be an efficient method for generating fine-structure human genetic maps.

Alleles↗
PubMed1989

Human immunodeficiency viral DNA is readily found in lymph node biopsies from seropositive individuals. Analysis of fixed tissue using the polymerase chain reaction.

Human immunodeficiency virus (HIV) DNA was detected in formalin-fixed, paraffin-embedded lymph node biopsies after in vitro DNA amplification by the polymerase chain reaction. Twenty-three of 25 biopsies from HIV seropositive individuals were positive for HIV DNA including 11 with follicular hyperplasia, six with follicular involution, two who were partially involved with Kaposi's sarcoma, one with granulomatous lymphadenitis, and three with non-Hodgkin's lymphoma. The remaining two biopsies from seropositive individuals lacking detectable HIV DNA also contained non-Hodgkin's lymphomas. An average of 0.0001 to 0.01 HIV DNA copies per cell was estimated to be present in biopsies with follicular hyperplasia or involution. The positive lymphoma biopsies contained approximately tenfold fewer HIV DNA. In contrast, 19 of 20 biopsies from seronegative or low risk individuals were negative for HIV DNA. The sole exception was a seronegative individual with chronic adenopathy from follicular hyperplasia and a history of prostitute contact. The studies demonstrated a high prevalence of HIV DNA in non-lymphomatous lymph node biopsies from HIV infected individuals.

Base Sequence↗
PubMed1988

Analysis of DNA sequences in forty-year-old paraffin-embedded thin-tissue sections: a bridge between molecular biology and classical histology.

DNA sequences from human tissues paraffin embedded 40 yr ago were studied using the in vitro gene amplification technique known as the polymerase chain reaction. Although significant DNA degradation was observed, single copy genomic sequences and viral segments were readily detected from single 5- to 10-microns tissue sections. This demonstrates that the world-wide collection of archival paraffin-embedded tissues may be used to study the association of biological agents (viral, bacterial, or parasitic) or endogenous DNA lesions with disease over time and to carry out retrospective studies on material where the clinical outcome has already been established. This will be especially valuable in studying rare cancers and other rare diseases.

Base Sequence↗
PubMed1988

Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes.

Using in vitro gene amplification by the polymerase chain reaction (PCR) and mutation detection by the RNAase A mismatch cleavage method, we have examined c-K-ras genes in human pancreatic carcinomas. We used frozen tumor specimens and single 5 micron sections from formalin-fixed, paraffin-embedded tumor tissue surgically removed or obtained at autopsy. Twenty-one out of 22 carcinomas of the exocrine pancreas contained c-K-ras genes with mutations at codon 12. In seven cases tested, the mutation was present in both primary tumors and their corresponding metastases. No mutations were detected in normal tissue from the same cancer patients or in five gall bladder carcinomas. We conclude from these results that c-K-ras somatic mutational activation is a critical event in the oncogenesis of most, if not all, human cancers of the exocrine pancreas.

Adenocarcinoma↗
PubMed1988

Detection of human papilloma virus in paraffin-embedded tissue using the polymerase chain reaction.

Human papilloma virus (HPV) DNA sequences have been detected in paraffin-embedded tissue using an enzymatic in vitro amplification technique known as the polymerase chain reaction. Amplification of a HPV DNA sequence before its detection with a cDNA probe significantly increases the rapidity as well as the sensitivity of detection such that a single 5-10-micron thick paraffin-embedded tissue section can be analyzed within 24 h. The assay specifically detected HPV 16 or 18 without crossreactivity with HPV 6 or 11. As few as 20 viral copies could be detected. The rapid and sensitive analysis of HPV in normal and pathological tissues using this technique may contribute significantly to identifying the role of HPV as a risk factor in carcinoma.

Carcinoma, Squamous Cell↗
PubMed1988

Linkage disequilibrium in human ribosomal genes: implications for multigene family evolution.

Members of the rDNA multigene family within a species do not evolve independently, rather, they evolve together in a concerted fashion. Between species, however, each multigene family does evolve independently indicating that mechanisms exist which will amplify and fix new mutations both within populations and within species. In order to evaluate the possible mechanisms by which mutation, amplification and fixation occur we have determined the level of linkage disequilibrium between two polymorphic sites in human ribosomal genes in five racial groups and among individuals within two of these groups. The marked linkage disequilibrium we observe within individuals suggests that sister chromatid exchanges are much more important than homologous or nonhomologous recombination events in the concerted evolution of the rDNA family and further that recent models of molecular drive may not apply to the evolution of the rDNA multigene family.

Biological Evolution↗
PubMed1988

Detection of cytomegalovirus DNA in peripheral blood of patients infected with human immunodeficiency virus.

Detection of cytomegalovirus (CMV) DNA can be facilitated by the polymerase chain reaction (PCR), an in vitro gene amplification technique. Twenty-eight CMV tissue culture isolates were examined by amplification of two separate CMV genes. All were found to contain CMV, although two of the isolates were positive for only one of the two genes. No detectable amplification occurred with human genomic or other viral DNA controls. The amplification products from as few as one CMV plaque-forming unit could be detected after the PCR. CMV DNA was detected in the blood of 14 of 27 patients with AIDS and one of six patients who were infected with human immunodeficiency virus but who did not have AIDS. Normal CMV-seropositive or -seronegative individuals did not have CMV DNA detected in their blood. The CMV PCR was more sensitive than the standard culture assay, can be completed in one to two days, uses only 20 microL of blood, and may be useful for rapidly detecting CMV in clinical specimens.

Acquired Immunodeficiency Syndrome↗
PubMed1988

Detection of human papillomavirus in normal and dysplastic tissue by the polymerase chain reaction.

Detection of human papilloma virus (HPV) types 16 and 18 in formalin-fixed, paraffin-embedded tissue by a new in vitro DNA amplification method, the polymerase chain reaction, was compared with detection with genomic DNA probes using in situ hybridization. The polymerase chain reaction replicates exponentially HPV DNA sequences present in a single 5- to 10-micron paraffin-embedded tissue section. The amplified sequences are detected with a DNA hybridization probe in a dot blot assay. The HPV polymerase chain reaction was able to detect on the average less than one HPV genome/cell as determined by tests of paraffin sections of cell pellets with known HPV genomic content. Cervical sections from 21 patients with HPV types 16, 18, or 31 as determined by in situ DNA hybridization were analyzed by the polymerase chain reaction. No disagreements between the two methods were detected. The sections comprising normal and dysplastic epithelium were further analyzed by the HPV polymerase chain reaction. The presence of virus correlated with the presence of dysplasia in the sections, though 3 of 10 normal sections contained HPV, and 1 of 21 sections with dysplasia lacked HPV 16 or 18. The polymerase chain reaction can specifically detect HPV 16 or 18 with high sensitivity from paraffin-embedded tissues.

Autoradiography↗
PubMed1987

Rapid prenatal diagnosis of sickle cell anemia by a new method of DNA analysis.

We have used a new method of DNA analysis for the rapid prenatal diagnosis of sickle cell anemia in two fetuses at risk for this disease. This method of detecting the sickle gene is a modification of standard restriction-enzyme techniques and requires only a small amount of DNA. The first step involves a 200,000-fold enzymatic amplification of the specific beta-globin DNA sequences that may carry the sickle mutation. This provides a sufficient quantity of DNA for the analysis. Next, a short radiolabeled synthetic DNA sequence homologous to normal beta A-globin gene sequences is hybridized to the amplified target sequences. The hybrid "duplexes" are then digested sequentially with two restriction endonucleases. The presence of beta A- or beta S-globin gene sequences in the amplified target DNA from the patient determines whether the beta A-hybridization probe anneals perfectly or with a single nucleotide mismatch. This difference affects the restriction-enzyme digestion of the DNA and the size of the resulting radiolabeled digestion products, which can be distinguished by electrophoresis followed by autoradiography. This method is sufficiently sensitive and rapid that the prenatal diagnosis of sickle cell anemia can be made on the same day that the fetal DNA is made available. It can also be applied to the diagnosis of hemoglobin C disease.

Anemia, Sickle Cell↗