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N Arnheim

Publications and source records attributed to N Arnheim.

At least 19 recordsLinked to original sources
PubMed1992

Extension of base mispairs by Taq DNA polymerase: implications for single nucleotide discrimination in PCR.

Thermus aquaticus (Taq) DNA polymerase was used to measure the extension efficiency for all configurations of matched and mismatched base pairs at template-primer 3'-termini. The transition mispairs, A(primer).C, C.A, G.T, and T.G were extended 10(-3) to 10(-4)-fold less efficiently than their correctly paired counterparts. Relative efficiencies for extending transversion mispairs were 10(-4) to 10(-5) for T.C and T.T, about 10(-6) for A.A, and less than 10(-6) for G.A, A.G, G.G and C.C. The transversion mispair C(primer).T was extended with high efficiency, about 10(-2) compared to a correct A.T basepair. The unexpected ease of extending the C.T mismatch was not likely to have been caused by primer-template misalignment. Taq polymerase was observed to bind with similar affinities to each of the correctly paired and mispaired primer-template 3'-ends. Thus, the failure of Taq polymerase to extend mismatches efficiently appears to be an intrinsic property of the enzyme and not due to an inability to bind to 3'-terminal mispairs. For almost all of the mispairs, C.T being the exception, Taq polymerase exhibits about 100 to 1000-fold greater discrimination against mismatch extension compared to avian myeloblastosis reverse transcriptase and HIV-1 reverse transcriptase which extend most mismatched basepairs permissively. Relative mismatch extension efficiencies for Taq polymerase were measured at 45 degrees C, 55 degrees C and 70 degrees C and found to be independent of temperature. The mispair extension data should be important in designing experiments using PCR to distinguish between sequences that vary by a single nucleotide.

Base Composition↗
PubMed1992

A pattern of accumulation of a somatic deletion of mitochondrial DNA in aging human tissues.

An assay that selectively amplifies a specific deletion of the mitochondrial genome has been used to study the extent of the deletion's accumulation in a variety of human tissues. The deletion occurs at much higher levels in nervous and muscle tissues than in all other tissues studied. The variation in deletion level between the same tissues in different persons of similar age appears to be less than the variation among tissues within an individual. Tests for artifactual explanations of the level differences were each negative. Three cellular parameters that are correlated with the level of the deletion are identified. The preferential accumulation of deleterious mitochondrial mutations in a restricted subset of aging human tissues may compound deficiencies of function in those tissues that accrue with age.

Adolescent↗
PubMed1992

Whole genome amplification from a single cell: implications for genetic analysis.

We have developed an in vitro method for amplifying a large fraction of the DNA sequences present in a single haploid cell by repeated primer extensions using a mixture of 15-base random oligonucleotides. We studied 12 genetic loci and estimate that the probability of amplifying any sequence in the genome to a minimum of 30 copies is not less than 0.78 (95% confidence). Whole genome amplification beginning with a single cell, or other samples with very small amounts of DNA, has significant implications for multipoint mapping by sperm or oocyte typing and possibly for genetic disease diagnosis, forensics, and the analysis of ancient DNA samples.

Base Sequence↗
PubMed1992

Base mispair extension kinetics. Binding of avian myeloblastosis reverse transcriptase to matched and mismatched base pair termini.

We investigate the enzymatic basis for the inefficient extension of single base mismatches by DNA polymerase compared with the extension of correct base pairs. Inefficient mismatch extension could result from either a reduced binding of the enzyme to mispaired versus correctly paired DNA template-primer termini, or from a lowered intrinsic rate of extension of mispairs by a bound enzyme, or from a combination of both factors. Avian myeloblastosis reverse transcriptase is used to measure the affinities (equilibrium dissociation constants) for the four matched and twelve mismatched base pair configurations situated at a primer 3'-terminus. The binding affinities are analyzed by two different assays employing polyacrylamide gels. The first assay uses steady-state kinetics to measure the efficiency of elongating correct and incorrect base pairs and to evaluate the enzyme's dissociation constants for matched and mismatched termini. The estimated KD values obtained in the steady-state analysis fall within a range of approximately 0.1-20 nM. The efficiencies of extending two of the mispairs, G.G and C.C, are too low to allow a determination of KD by the kinetics method. The second assay uses equilibrium binding to measure the ratio of polymerase bound to matched compared with mismatched termini, KDright/KDwrong. The affinity ratios, including values for G.G and C.C mispairs, are in the range of about 0.4-4.2. While around 1 order of magnitude difference is observed in the relative binding affinities of the polymerase for matched and mismatched primer termini, the relative extension efficiencies vary over more than 5 orders of magnitude. Therefore, it appears that inefficient mismatch extension is caused primarily by a kinetic block inhibiting elongation from mispaired primer 3'-termini rather than to a difference in binding.

Avian Myeloblastosis Virus↗
PubMed1992

Using the polymerase chain reaction to estimate mutation frequencies and rates in human cells.

The Polymerase Chain Reaction (PCR) has had a significant impact on molecular studies of human mutagenesis, mainly in the acceleration of molecular characterisation of mutant genes in cells isolated by a phenotypic selection. PCR can also be used to study genetic alterations in cells which have not undergone phenotypic selection. By modifying the standard PCR parameters, the presence of mutations can be assayed in single human cells, creating the potential to determine mutation rates in gametes on a cell-by-cell basis (Section I). Alternatively, PCR can be used to selectively amplify a mutant gene in a pool of normal genomes and thus determine a mutation frequency (Section II). Current applications of these two approaches are summarised and critically reviewed.

Animals↗
PubMed1992

Gene-centromere linkage mapping by PCR analysis of individual oocytes.

We describe a general method of determining the recombination fraction between a polymorphic locus and the centromere in any species where single oocytes can be obtained. After removal of the first polar body, each oocyte is analyzed by PCR. The frequency of oocytes heterozygous at the polymorphic locus is used to estimate the recombination fraction. We estimate a recombination fraction of 0.15 between the mouse major histocompatibility complex (H-2) and the centromere of chromosome 17.

Animals↗
PubMed1992

Close linkage between bovine prolactin and BoLA-DRB3 genes: genetic mapping in cattle by single sperm typing.

The major histocompatibility complex and prolactin (PRL) genes are syntenic in humans and cattle but the genetic distance between these loci has not been determined for either species. In this study, the sperm typing technique was used to measure the recombination frequency between the bovine lymphocyte antigen (BoLA)-DRB3 and PRL loci. A total of 300 sperm were typed from one doubly heterozygous bull for segregation of DRB3 and PRL alleles. Sperm typing was performed using the polymerase chain reaction (PCR) and restriction enzyme cleavage of the PCR products, followed by resolution of the restriction fragments in polyacrylamide gels. Digestion with the restriction endonuclease RsaI allowed the unambiguous discrimination of alleles for both loci. The maximum likelihood estimation of the recombination fraction theta = 0.04, with a 95% confidence interval of 0.01 to 0.07. Close linkage between PRL and DRB3 has important implications for marker-assisted selection in animal breeding since PRL has been shown to be closely linked to a locus that affects milk yield, and BoLA loci influence susceptibility to a number of infectious diseases. Our results demonstrate the general applicability of the sperm typing procedure for gene mapping in species other than humans and provide an example of how parallel efforts to map the genomes of agriculturally important species of animals can have a positive impact on the development of a primary human linkage map.

Alleles↗
PubMed1992

Sperm typing allows accurate measurement of the recombination fraction between D3S2 and D3S3 on the short arm of human chromosome 3.

The interval between the D3S2 and D3S3 loci on human chromosome 3p is a frequent site of deletions in a number of different cancers and contains the most common fragile site in man. Both loci have been physically mapped to 3p but because heterozygosity for D3S3 is so infrequent, recombination between them could not be determined accurately by using family studies. Sperm typing can measure recombination between DNA polymorphisms even in a single individual and thus can make use of polymorphisms with a low PIC. The recombination fraction between D3S2 and D3S3 was estimated to be 0.28 based on analyzing 189 and 77 sperm from two doubly heterozygous donors, respectively. These results demonstrate one of the ways in which sperm typing can complement pedigree analysis in constructing genetic maps.

Base Sequence↗
PubMed1992

Deleterious mitochondrial DNA mutations accumulate in aging human tissues.

This paper reviews the current state of knowledge of the contribution of mitochondrial DNA (mtDNA) mutations to the phenotype of aging. Its major focus is on the discovery of deletions of mtDNA which previously were thought to occur only in individuals with neuromuscular disease. One particular deletion (mtDNA4977) accumulates with age primarily in non-dividing cells such as muscle and brain of normal individuals. The level of the deletion rises with age by more than 1000 fold in heart and brain and to a lesser extent in other tissues. In the brain, different regions have substantially different levels of the deletion. High levels of accumulation of the deletion in tissues are correlated with high oxygen consumption. We speculate that oxidative damage to mtDNA may be 'catastrophic'; mutations affecting mitochondrially encoded polypeptides involved in electron transport could increase free radical generation leading to more mtDNA damage.

Aging↗
PubMed1992

Mosaicism for a specific somatic mitochondrial DNA mutation in adult human brain.

The levels of a specific mitochondrial DNA deletion (mtDNA4977) measured in 12 brain regions of 6 normal adults 39 to 82 years old exhibited striking variation among anatomical locations. Comparisons of the same region among individuals showed an increase of mtDNA4977 with age. The three regions with the highest levels, caudate, putamen and substantia nigra, are characterized by a high dopamine metabolism. The breakdown of dopamine by mitochondrial MAO produces H2O2 which can lead to oxygen radical formation. We suggest that mtDNA4977 may be the "tip of the iceberg" of the spectrum of somatic mutations produced by oxidative damage.

Adult↗
PubMed1992

Genetic analysis using the polymerase chain reaction.

The ability of PCR to amplify a specific DNA segment from a complex template in an automated reaction has made molecular analysis more accessible both to basic research and diagnostic laboratories. Modifications of the basic PCR method using generic primers now allow DNA amplification even in the absence of specific nucleotide sequence information. Although PCR has proved extremely valuable for detecting the presence or absence of a given sequence (e.g. diagnosis of infectious disease pathogens like HIV), it is uniquely suited to analyze genetic variation in the amplified DNA. The capacity to analyze DNA variation from limited samples like hair or cheek scrapings has made genetic diagnosis and population genetics studies much simpler and the amplification of specific sequences from individual cells (e.g. sperm) has opened new approaches to genetic mapping.

Biological Evolution↗
PubMed1992

A new source of polymorphic DNA markers for sperm typing: analysis of microsatellite repeats in single cells.

We show that dinucleotide and tetranucleotide repeat polymorphisms can be analyzed in single cells without using radioactivity or denaturing gels. This provides a rich new source of DNA polymorphisms for genetic mapping by sperm typing. The recombination fraction between two CA repeat polymorphisms was determined after whole genome amplification of single sperm, followed by typing of two different aliquots, one aliquot for each polymorphic locus. Single-cell analysis of microsatellites may also be valuable both for preimplantation genetic disease diagnosis based on single-blastomere or polar-body analysis and for the typing of forensic or ancient DNA samples containing very small amounts of nucleic acid.

Apolipoprotein C-II↗
PubMed1992

A multiple-tubes approach for accurate genotyping of very small DNA samples by using PCR: statistical considerations.

A multiple-tubes procedure is described for using PCR to determine the genotype of a very small DNA sample. The procedure involves dividing the sample among several tubes, then amplifying and typing the contents of each tube separately. The results are analyzed by a statistical procedure which determines whether a genotype can be conclusively assigned to the DNA sample. Simulation studies show that this procedure usually gives correct results even when the number of double-stranded fragments in the sample is as small as 30. The procedure remains effective even in the presence of small amounts of laboratory contamination. We find that the multiple-tubes procedure is superior to the standard one-tube procedure, either when the sample is small or when laboratory contamination is a potential problem; and we recommend its use in these situations. Because the procedure is statistical, it allows the degree of certainty in the result to be quantified and may be useful in other PCR applications as well.

DNA↗
PubMed1991

Eliminating primers from completed polymerase chain reactions with exonuclease VII.

Single-stranded oligonucleotide primers can be efficiently removed after PCR using E.coli exonuclease VII. Even only a few molecules of double stranded PCR product are unaffected by a treatment which eliminates 20 picomoles of primer in the presence of 500 ng of denatured genomic DNA. Exonuclease VII treatment is rapid and could simplify complicated multistep PCR protocols.

Electrophoresis, Agar Gel↗