PubMed Health⌕ Search

Biomedical subjects

John M Butler

Publications and source records attributed to John M Butler.

33 records · Page 2Linked to original sources
PubMed2004

High-throughput Y-STR typing of U.S. populations with 27 regions of the Y chromosome using two multiplex PCR assays.

Two Y-chromosome short tandem repeat (STR) multiplex polymerase chain reaction (PCR) assays were used to generate haplotypes for 19 single copy and 3 multi-copy Y-STRs. A total of 27 PCR products were examined in each sample using the following loci: DYS19, DYS385 a/b, DYS388, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS426, DYS437, DYS438, DYS439, DYS447, DYS448, DYS450, DYS456, DYS458, DYS460, DYS464 a/b/c/d, H4, and YCAII a/b. The first multiplex is the Y-STR 20plex previously described by Butler et al. [Forensic Sci. Int. 129 (2002) 10]. The second multiplex is a novel Y-STR 11plex and includes DYS385 a/b, DYS447, DYS448 and the new markers DYS450, DYS456, DYS458, and DYS464 a/b/c/d. These two multiplexes were tested on 647 males from three United States population sample sets: 260 African Americans, 244 Caucasians, and 143 Hispanics. Haplotype comparisons between common loci included in the 20plex and 11plex assays as well as commercially available kits found excellent agreement across a sampling of the population samples. The multi-copy loci DYS464, DYS385, and YCAII were the most polymorphic followed by the following single copy Y-STRs: DYS458, DYS390, DYS447, DYS389II, DYS448, and DYS456. Samples containing the most common type in the European database could be well resolved with additional markers beyond the minimal haplotype loci.

Alleles↗
PubMed2004

Forensic DNA typing by capillary electrophoresis using the ABI Prism 310 and 3100 genetic analyzers for STR analysis.

DNA typing with short tandem repeat (STR) markers is now widely used for a variety of applications including human identification. Capillary electrophoresis (CE) instruments, such as the ABI Prism 310 and ABI 3100 Genetic Analyzers, are the method of choice for many laboratories performing STR analysis. This review discusses issues surrounding sample preparation, injection, separation, detection, and interpretation of STR results using CE systems. Requirements for accurate typing of STR alleles are considered in the context of what future analysis platforms will need to increase sample throughput and ease of use.

Electrophoresis, Capillary↗
PubMed2004

AutoDimer: a screening tool for primer-dimer and hairpin structures.

The ability to select short DNA oligonucleotide sequences capable of binding solely to their intended target is of great importance in developing nucleic acid based detection technologies. Applications such as multiplex PCR rely on primers binding to unique regions in a genome. Competing side reactions with other primer pairs or template DNA decrease PCR efficiency: Freely available primer design software such as Primer3 screens for potential hairpin and primer-dimer interactions while selecting a single primer pair. The development of multiplex PCR assays (in the range of 5 to 20 loci) requires the screening of all primer pairs for potential cross-reactivity. However, a logistical problem results due to the number of total number of comparisons required. Comparing the primer set for a 10-plex assay (20 total primer sequences) results in 210 primer-primer combinations that must be screened. The ability to screen sets of candidate oligomers rapidly for potential cross-reactivity reduces overall assay devlelopment time. Here we report the application of a familiar sliding algorithm for comparing two strands of DNA in an overlapping fashion. The algorithm has been employed in a software package wherein the user can compare multiple sequences in a single computational run. After the screening is completed, a score is assigned to potential duplex interactions exceeding a user-defined threshold. Additional criteria of predicted melting temperature (Tm) and free energy of melting (deltaG) are included for further ranking. Sodium counterion and total stand concentrations can be adjusted for the Tm and deltaG calculations. The predicted interactions are saved in a text file for further evaluation.

Algorithms↗
PubMed2004

Y-SNP typing of U.S. African American and Caucasian samples using allele-specific hybridization and primer extension.

Multiplex analysis of genetic markers has become increasingly important in a number of fields, including DNA diagnostics and human identity testing. Two methods for examination of single nucleotide polymorphisms (SNPs) with a potential for a high degree of multiplex analysis of markers are primer extension with fluorescence detection, and allele-specific hybridization using flow cytometry. In this paper, we examined 50 different SNPs on the Y-chromosome using three primer extension multiplexes and five hybridization multiplex assays. For certain loci, the allele-specific hybridization method exhibited sizable background signal from the absent alternate allele. However, 100% concordance (>2000 alleles) was observed in ten markers that were typed using both methods. A total of 18 unique haplogroups out of a possible 45 were observed in a group of 229 U.S. African American and Caucasian males with the majority of samples being assigned into 2 of the 18 haplogroups.

Alleles↗
PubMed2004

A study on the effects of degradation and template concentration on the amplification efficiency of the STR Miniplex primer sets.

In forensic DNA analysis, the samples recovered from the crime scene are often highly degraded leading to poor PCR amplification of the larger sized STR loci. To avoid this problem, we have developed STR markers with redesigned primer sequences called "Miniplexes" to produce smaller amplicons. To assess the effectiveness of these kits, we have tested these primer sets with enzymatically degraded DNA and compared the amplifications to a commercial kit. We also conducted sensitivity and peak balance studies of three Miniplex sets. Lastly, we report a case study on two human skeletal remain samples collected from different environmental conditions. In both types of degraded DNA, the Miniplex primer sets were capable of producing more complete profiles when compared to the larger sized amplicons from the commercial kit. Correct genotypes were obtained at template concentrations as low as 31 pg/25 microL. Overall, our data confirm that our redesigned primers can increase the probability of obtaining a usable profile in situations where standard kits fail.

DNA Damage↗
PubMed2003

NIST Mixed Stain Study 3: DNA quantitation accuracy and its influence on short tandem repeat multiplex signal intensity.

The Mixed Stain Study 3 (MSS3) interlaboratory challenge exercise evaluated the 2001 performance of STR multiplex DNA typing systems using a set of seven DNA extracts of designed concentration and composition. This initial report focuses on the linkages connecting the measurement of the concentration of DNA ([DNA]) to the observed STR multiplex signal intensities. There is a causal relationship between [DNA] measurement accuracy and the efficiency of STR multiplex analysis. There are no intrinsic measurement performance differences among the [DNA] measurement technologies reported. However, there are large differences in the efficiencies of amplification, separation, and detection among participants using the same nominal measurement systems.

DNA↗
PubMed2003

17 beta-Oestradiol attenuates nucleotide excision repair.

Epidemiological studies strongly suggest associations between chronic exposure to endogenous oestrogens and the development of breast and gynaecological tumours. Two mechanisms by which 17 beta-oestradiol (E2) may enhance tumorigenesis are: (i) enhancement of cell proliferation and (ii) the production of reactive, genotoxic metabolites. Here we suggest an additional mechanism, inhibition of DNA repair. The removal of UV-induced thymine dimers from human keratinocytes, reflective of nucleotide excision repair, was significantly attenuated by treatment of cells with E2. In contrast, treatment with 17 alpha-oestradiol had no effect. Mechanisms are proposed for this effect of E2, which may contribute to its carcinogenic potential.

Cell Line↗
PubMed2003

Multiplex PCR design strategy used for the simultaneous amplification of 10 Y chromosome short tandem repeat (STR) loci.

The simultaneous amplification of multiple regions of a DNA template is routinely performed using the polymerase chain reaction (PCR) in a process termed multiplex PCR. A useful strategy involving the design, testing, and optimization of multiplex PCR primer mixtures will be presented. Other multiplex design protocols have focused on the testing and optimization of primers, or the use of chimeric primers. The design of primers, through the close examination of predicted DNA oligomer melting temperatures ( T(m)) and primer-dimer interactions, can reduce the amount of testing and optimization required to obtain a well-balanced set of amplicons. The testing and optimization of the multiplex PCR primer mixture constructed here revolves around varying the primer concentrations rather than testing multiple primer combinations. By solely adjusting primer concentrations, a well-balanced set of amplicons should result if the primers were designed properly. As a model system to illustrate this multiplex design protocol, a 10-loci multiplex (10plex) Y chromosome short tandem repeat (STR) assay is used.

Base Sequence↗
PubMed2003

The development of reduced size STR amplicons as tools for analysis of degraded DNA.

New multiplex PCR sets of commonly used short tandem repeat (STR) markers have been developed to produce PCR products that are reduced in size when compared to standard commercial STR kits. The reduction in size of these amplicons can facilitate the examination and analysis of degraded DNA evidence by improving amplification efficiency. This "miniSTR" approach will permit current forensic practitioners to use STR markers and instrumentation already present in their laboratories and to generate genotyping data that is directly comparable to reference samples and searchable through the FBI's Combined DNA Index System (CODIS) databases. This paper discusses the development of these new primer sets and presents some initial results in the analysis of degraded and aged DNA samples. A method for removal of problematic fluorescent dye artifacts is also described. Comparison studies in over 100 samples have verified that these miniSTR primers can provide fully concordant results to commercial STR kits and can provide improved signal from degraded DNA specimens. These miniplex sets should prove valuable in the analysis of samples where allele dropout and reduced sensitivity of larger STR alleles occurs.

Blotting, Southern↗
PubMed2002

Forensic value of 14 novel STRs on the human Y chromosome.

We identified and characterized 14 novel short-tandem-repeats (STRs) on the Y chromosome and typed them in two samples, a globally diverse panel of 73 cell lines, and 148 individuals from a European-American population. These Y-STRs include eight tetranucleotide repeats (DYS449, DYS453, DYS454, DYS455, DYS456, DYS458, DYS459, and DYS464), five pentanucleotide repeats (DYS446, DYS447, DYS450, DYS452, and DYS463), and one hexanucleotide repeat (DYS448). Sequence data were obtained to designate a repeat number nomenclature. The gene diversities of an additional 22 Y-STRs, including the most commonly used in forensic databases, were directly compared in the cell line DNAs. Six of the 10 most polymorphic markers include the newly identified Y-STRs. Furthermore, these novel Y-STRs greatly improved the resolution of paternal lineages, above the level obtained with commonly used Y-STRs, in the European-American population.

Alleles↗
PubMed2002

A novel multiplex for simultaneous amplification of 20 Y chromosome STR markers.

A multiplex polymerase chain reaction (PCR) assay capable of simultaneously amplifying 20 Y chromosome short tandem repeat (STR) markers has been developed to aid human identity testing and male population studies. These markers include all of the Y STRs that make up the "extended haplotype" used in Europe (DYS19, DYS385, DYS389I/II, DYS390, DYS391, DYS392, DYS393, and YCAII) plus additional polymorphic Y STRs (DYS437, DYS438, DYS439, DYS447, DYS448, DYS388, DYS426, GATA A7.1, and GATA H4). Primers for the markers DYS385, DYS389, and YCAII target duplicated regions of the Y chromosome and thus can provide two polymorphic peaks for each respective primer set. This Y STR 20plex, which utilizes 34 different PCR primers, is the first to include a simultaneous amplification of all the markers within the European "minimal" and "extended" haplotypes. Relative primer positions are compared between the newly developed primers described here and previously published ones. Efforts were made to avoid X chromosome homology in the primer design as well as close packing of PCR product size ranges in order to keep all alleles less than 350 bp through careful examination of known allele ranges. Haplotype comparisons between the 20plex and a commercially available kit found excellent agreement across the 76 samples in the Y chromosome consortium panel.

Alleles↗
PubMed2002

A strategy for examining complex mixtures of deoxyoligonucleotides using ion-pair-reverse-phase high-performance liquid chromatography, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and informatics.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and ion-pair-reverse-phase high-performance liquid chromatography (IP-RP HPLC) techniques were combined to determine the sequence identity of short single-stranded deoxyoligonucleotides. This methodology is demonstrated using a commercially available multiplex set of eight primer pairs. The primer pairs were separated and collected by IP-RP HPLC. Partial sequence information for IP-RP HPLC fractions was obtained from analyzing exonuclease digestion products by MALDI-TOF MS. IP-RP HPLC, MALDI-TOF MS, exonuclease digests, and a simple computational algorithm provide an integrated strategy for determining the sequence of short nucleic acid oligomers.

Algorithms↗
PubMed2002

Polymerase chain reaction amplification of DNA from aged blood stains: quantitative evaluation of the "suitability for purpose" of four filter papers as archival media.

In collaboration with the Armed Forces Institute of Pathology's Department of Defense DNA Registry, the National Institute of Standards and Technology recently evaluated the performance of a short tandem repeat multiplex with dried whole blood stains on four different commercially available identification card matrixes. DNA from 70 stains that had been stored for 19 months at ambient temperature was extracted or directly amplified and then processed using routine methods. All four storage media provided fully typeable (qualitatively identical) samples. After standardization, the average among-locus fluorescence intensity (electropherographic peak height or area) provided a suitable metric for quantitative analysis of the relative amounts of amplifiable DNA in an archived sample. The amounts of DNA in Chelex extracts from stains on two untreated high-purity cotton linter pulp papers and a paper treated with a DNA-binding coating were essentially identical. Average intensities for the aqueous extracts from a paper treated with a DNA-releasing coating were somewhat lower but also somewhat less variable than for the Chelex extracts. Average intensities of directly amplified punches of the DNA-binding paper were much larger but somewhat more variable than the Chelex extracts. Approximately 25% of the observed variation among the intensity measurements is shared among the four media and thus can be attributed to intrinsic variation in white blood count among the donors. All of the evaluated media adequately "bank" forensically useful DNA in well-dried whole blood stains for at least 19 months at ambient temperature.

Blood Stains↗