Large Amplicons Research Articles

Integrity of nuclear genomic deoxyribonucleic acid in cooked meat: Implications for food traceability

It is essential to isolate high-quality DNA from muscle tissue for PCR-based applications in traceability of animal origin. We wished to examine the impact of cooking meat to a range of core temperatures on the quality and quantity of subsequently isolated genomic (specifically, nuclear) DNA. Triplicate steak samples were cooked in a water bath (100 degrees C) until their final internal temperature was 75, 80, 85, 90, 95, or 100 degrees C, and DNA was extracted. Deoxyribonucleic acid quantity was significantly reduced in cooked meat samples compared with raw (6.5 vs. 56.6 ng/microL; P < 0.001), but there was no relationship with cooking temperature. Quality (A(260)/A(280), i.e., absorbance at 260 and 280 nm) was also affected by cooking (P < 0.001). For all 3 genes, large PCR amplicons (product size >800 bp) were observed only when using DNA from raw meat and steak cooked to lower core temperatures. Small amplicons (<200 bp) were present for all core temperatures. Cooking meat to high temperatures thus resulted in a reduced overall yield and probable fragmentation of DNA to sizes less than 800 bp. Although nuclear DNA is preferable to mitochondrial DNA for food authentication, it is less abundant, and results suggest that analyses should be designed to use small amplicon sizes for meat cooked to high core temperatures.

The effects of temperature on detection of prey DNA in two species of carabid beetle

PCR-based techniques to investigate predator-prey trophic interactions are starting to be used more widely, but factors affecting DNA decay in predator guts are still poorly understood. Here, we investigated the effects of time since feeding, temperature and amplicon size on the detectability of prey DNA in the gut content of two closely related predator species. Cereal aphids, Sitobion avenae, were fed to the carabid beetles Pterostichus melanarius and Nebria brevicollis. Beetles were allowed to digest their meal at 12 degrees C, 16 degrees C and 20 degrees C, and batches of beetles were subsequently frozen at time periods from 0-72 h after feeding. Aphid DNA was detected within beetles' gut contents using primers amplifying fragments of 85, 231, 317 and 383 bp. Prey DNA detection rates were significantly higher in N. brevicollis than in P. melanarius, indicating fundamental dissimilarities in prey digestion capacities. High temperatures (20 degrees C) and large amplicons (383 bp) significantly decreased detection rates. The shortest amplicon gave the highest prey DNA detection success, whereas no differences were observed between the 231 bp and the 317 bp fragment. Our results indicate that factors such as ambient temperature, predator taxon and amplicon size should all be considered when interpreting data derived from PCR-based prey detection. Correction for such factors should make calculation of predation rates in the field more accurate and could help us to estimate when predation events occur in the field.

Amplification at 7q22 Targets CDK6 in T-Cell Lymphoma.

Genomic amplification is microscopically visible extrachromosomally as double-minute chromosomes (DMIN) or intrachromosomally as homogeneously stained regions (HSR). Both DMIN and HSR comprise iterated copies of discrete genomic regions (amplicons) effecting copy number increases associated with upregulation of target genes. In cancer cells, these usually comprise oncogenes such as MYC. Cytogenetic analysis of the anaplastic large cell lymphoma (ALCL) cell line SU-DHL-1 revealed an HSR on the long arm region of chromosome 7 (7q). Detailled analysis of this region by fluorescence in situ hybridization (FISH) using tilepath BAC clones identified an amplicon corresponding to 86–95 Mb (NCBI Build 36.1) at 7q22. Expression analysis using reverse transcription (RT)-PCR of candidate oncogenes mapped within the amplicon, comprising DBF4, SRI, AKAP9, GATAD1, CDK6 and PPP1R9A, highlighted cyclin-dependent kinase 6 (CDK6) as a plausible amplification target. With reference to other ALCL cell lines, including SR-786, lacking this amplicon, SU-DHL-1 displays upregulation of CDK6 at both the RNA and protein levels as indicated by semiquantitative RT-PCR, real-time quantitative (RQ)-PCR and immuno-cytochemistry. Another ALCL cell line, L-82, containing a larger amplicon (7q21-31), also showed enhanced CDK6 expression as analyzed by RQ-PCR. For a functional test both SU-DHL-1 and SR-786 cells were treated with rapamycin, an inhibitor of the AKT-pathway which is involved in degradation of CDK-inhibitor p27. In contrast to SR-786, SU-DHL-1 cells are resistant to the effects of rapamycin on both proliferation and G1 cell cycle arrest, implying that CDK6 overexpression may confer proliferative advantage. Copy number analysis of several tumor-relevant genes using a commercial PCR-based approach (MLPA) determined a nearly 5-fold amplification of the CDK6 gene in SU-DHL-1. Interphase FISH confirmed copy number increase in SU-DHL-1 and identified CDK6 amplification in 1/77 ALCL and 4/38 peripheral T-cell lymphoma patient samples, indicating that this genetic event is relatively rare among T-cell lymphomas. Taken together, we have identified an amplicon at 7q22 in T-cell lymphoma cells targeting CDK6 which is an important cell cycle regulator and probably connected to the increased proliferative capacity of these malignant cells. Although CDK6 amplification seems to be infrequent overall, it strengthens the importance of this gene in regulating proliferation among T-cell lymphomas and may, therefore, represent a potential molecular target for therapeutic intervention in these disease entities.

Closed-Tube SNP Genotyping Without Labeled Probes

Two methods for closed-tube single nucleotide polymorphism (SNP) genotyping without labeled probes have become available: unlabeled probe and amplicon melting. Unlabeled probe and amplicon melting assays were compared using 5 SNPs: human platelet antigens 1, 2, 5, and 15 and a C>T variant located 13910 base pairs (bp) upstream of the lactase gene. LCGreen Plus (Idaho Technology, Salt Lake City, UT) was used as the saturating DNA dye. Unlabeled probe data were readily interpretable and accurate for all amplicon lengths tested. Five targets that ranged in size from 42 to 72 bp were well resolved by amplicon melting on the LightScanner (Idaho Technology) or LightTyper (Roche, Indianapolis, IN) with no errors in genotyping. However, when larger amplicons (206 bp) were used and analyzed on lower resolution instruments (LightTyper and I-Cycler, Bio-Rad, Hercules, CA), the accuracy of amplicon genotyping was only 73% to 77%. When 2 temperature standards were used to bracket the amplicon of interest, the accuracy of amplicon genotyping of SNPs was increased to 100% (LightTyper) and 88% (I-Cycler).

High-resolution mapping identifies a commonly amplified 11q13.3 region containing multiple genes flanked by segmental duplications

DNA amplification of the 11q13 region is observed frequently in many carcinomas. Within the amplified region several candidate oncogenes have been mapped, including cyclin D1, TAOS1 and cortactin. Yet, it is unknown which gene(s) is/are responsible for the selective pressure enabling amplicon formation. This is probably due to the use of low-resolution detection methods. Furthermore, the size and structure of the amplified 11q13 region is complex and consists of multiple amplicon cores that differ between different tumor types. We set out to test whether the borders of the 11q13 amplicon are restricted to regions that enable DNA breakage and subsequent amplification. A high-resolution array of the 11q13 region was generated to study the structure of the 11q13 amplicon and analyzed 29 laryngeal and pharyngeal carcinomas and nine cell lines with 11q13 amplification. We found that boundaries of the commonly amplified region were restricted to four segments. Three boundaries coincided with a syntenic breakpoint. Such regions have been suggested to be putatively fragile. Sequence comparisons revealed that the amplicon was flanked by two large low copy repeats known as segmental duplications. These segmental duplications might be responsible for the typical structure and size of the 11q13 amplicon. We hypothesize that the selection for genes through amplification of the 11q13.3 region is determined by the ability to form DNA breaks within specific regions and, consequently, results in large amplicons containing multiple genes.

RCL2, a New Fixative, Preserves Morphology and Nucleic Acid Integrity in Paraffin-Embedded Breast Carcinoma and Microdissected Breast Tumor Cells

Methacarn and RCL2, a new noncrosslinking fixative, were compared to formalin-fixed or frozen tissue samples of the same invasive breast carcinoma and were evaluated for their effects on tissue morphology and immunohistochemistry as well as DNA and RNA integrity. The histomorphology of methacarn- or RCL2-fixed paraffin-embedded tumors was similar to that observed with the matched formalin-fixed tissues. Immunohistochemistry using various antibodies showed comparable results with either fixative, leading to accurate breast tumor diagnosis and determination of estrogen and progesterone receptors, and HER2 status. Methacarn and RCL2 fixation preserved DNA integrity as demonstrated by successful amplification and sequencing of large DNA amplicons. Similarly, high-quality RNA could be extracted from methacarn- or RCL2-fixed paraffin-embedded MCF-7 cells, whole breast tumor tissues, or microdissected breast tumor cells, as assessed by electropherogram profiles and real-time reverse transcriptase-polymerase chain reaction quantification of various genes. Moreover, tissue morphology and RNA integrity were preserved after 8 months of storage. Altogether, these results indicate that methacarn, as previously shown, and RCL2, a promising new fixative, have great potential for performing both morphological and molecular analyses on the same fixed tissue sample, even after laser-capture microdissection, and can open new doors for investigating small target lesions such as premalignant breast lesions.

Comparative Evaluation of Cleavase Fragment Length Polymorphism With PCR-SSCP and PCR-RFLP to Detect Antimicrobial Agent Resistance in Mycobacterium tuberculosis.

Background: Several molecular methods potentially useful in the detection of Mycobacterium tuberculosis mutations, specifically in rpoB and katG, were compared. Methods and Results: DNA from 24 M. tuberculosis clinical isolates, with mutations associated with resistance to rifampin and/or isoniazid, was analyzed. A 128 bp amplicon, spanning the 81 bp rpoB region containing most mutations leading to rifampin resistance, was analyzed by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and a recently introduced mutation scanning method, cleavase fragment length polymorphism (CFLP) analysis. Also, a 350 bp amplicon encompassing that region was analyzed by the CFLP method. CFLP analysis of the 350 bp amplicon (23 isolates) identified 14 of 17 mutants; however, CFLP analysis of the 128 bp amplicon accurately identified all mutants as did PCR-SSCP with interpretative difficulty for two codon 513 mutations. CFLP and PCR-restriction fragment length polymorphism (RFLP) analyses of a 623 bp amplicon encompassing katG codons 315 and 463 showed that the CFLP method identified single and dinucleotide codon 315 substitutions with or without codon 463 (CGG-->CTG) changes, whereas PCR-RFLP (MspI) missed one codon 315 polymorphism (AGC-->ACA) in three isolates. Conclusion: Both PCR-SSCP and CFLP analyses were sensitive in identifying all mutations on short sequences in the rpoB mutants. CFLP appears to be more efficient than SSCP and RFLP for the detection of mutations in large amplicons.

Targeted oligonucleotide-mediated microsatellite identification (TOMMI) from large-insert library clones

BackgroundIn the last few years, microsatellites have become the most popular molecular marker system and have intensively been applied in genome mapping, biodiversity and phylogeny studies of livestock. Compared to single nucleotide polymorphism (SNP) as another popular marker system, microsatellites reveal obvious advantages. They are multi-allelic, possibly more polymorphic and cheaper to genotype. Calculations showed that a multi-allelic marker system always has more power to detect Linkage Disequilibrium (LD) than does a di-allelic marker system [1]. Traditional isolation methods using partial genomic libraries are time-consuming and cost-intensive. In order to directly generate microsatellites from large-insert libraries a sequencing approach with repeat-containing oligonucleotides is introduced.ResultsSeventeen porcine microsatellite markers were isolated from eleven PAC clones by targeted oligonucleotide-mediated microsatellite identification (TOMMI), an improved efficient and rapid flanking sequence-based approach for the isolation of STS-markers. With the application of TOMMI, an average of 1.55 (CA/GT) microsatellites per PAC clone was identified. The number of alleles, allele size distribution, polymorphism information content (PIC), average heterozygosity (HT), and effective allele number (NE) for the STS-markers were calculated using a sampling of 336 unrelated animals representing fifteen pig breeds (nine European and six Chinese breeds). Sixteen of the microsatellite markers proved to be polymorphic (2 to 22 alleles) in this heterogeneous sampling. Most of the publicly available (porcine) microsatellite amplicons range from approximately 80 bp to 200 bp. Here, we attempted to utilize as much sequence information as possible to develop STS-markers with larger amplicons. Indeed, fourteen of the seventeen STS-marker amplicons have minimal allele sizes of at least 200 bp. Thus, most of the generated STS-markers can easily be integrated into multilocus assays covering a broader separation spectrum. Linkage mapping results of the markers indicate their potential immediate use in QTL studies to further dissect trait associated chromosomal regions.ConclusionThe sequencing strategy described in this study provides a targeted, inexpensive and fast method to develop microsatellites from large-insert libraries. It is well suited to generate polymorphic markers for selected chromosomal regions, contigs of overlapping clones and yields sufficient high quality sequence data to develop amplicons greater than 250 bases.

Confirmation of the Value of a Modified Long-distance Polymerase Chain Reaction in the Detection of Inversion Intron 22 in Severe Hemophilia A: A Technical Note

Hemophilia A is one of the most common bleeding disorders in man. Approximately half the families with a severe disease have an inversion of the factor VIII gene. The inversion may be detected with a long polymerase chain reaction. This is a simple and reproducible method that may yield satisfactory results in approximately 24 hours. The long-distance polymerase chain reaction amplify three very large amplicons with a very GC-rich region of several kilobases, and is detected with a Expand Long Template DNA polymerase (Roche, Mannheim, Germany). Recently this polymerase has been changed with a new chemical composition. The object of the present method is to standardize the technique using the new Expand Long polymerase. For the new protocol, the cycling conditions, the concentration of nucleotide primers, and the buffer are changed. The need for a rapid response is determined in the case of hemophilia A patients, not only by the desire to reach a proper classification, but also by the urgency to inform the carrier status of the mother or of a female relative.

Polymerase chain reaction inhibition assay documenting the amotosalen‐based photochemical pathogen inactivation process of platelet concentrates

The INTERCEPT Blood System (Baxter Healthcare Corp.) for platelets (PLTs) uses amotosalen-HCl (S-59) in conjunction with ultraviolet A (UVA) light to inactivate contaminating pathogens by modifying the nucleic acids of pathogens. The success of this photochemical treatment (PCT) process can be documented indirectly with a high-performance liquid chromatography assay measuring the photodegradation of amotosalen and measurement of the UVA light dose delivered by the illumination system. To develop an assay that documents the success of PCT directly on the effector molecule DNA, the effect of PCT on PLT-derived mitochondrial DNA (mtDNA) was examined. mtDNA-specific polymerase chain reaction (PCR) assays were tested with regard to their susceptibility for PCT, their reliability in terms of PCR performance, and the absence of polymorphic sites in primer hybridization loci. Suitable PCR amplification targets were found in the regions of 16S rDNA, cytochrome c oxidase I, and cytochrome c oxidase III of mitochondria. Amplicon sizes between 868 and 1248 bp gave consistent signals before PCT and complete inhibition of the PCR signal after PCT. Amplicons of less than 300 bp were found to be transparent to PCT. Based on PCT-mediated mtDNA modifications in PLTs, a PCR inhibition assay was established with a large amplicon documenting the success of PCT and a small amplicon serving as an internal control.

Quantitative Detection and Differentiation of Human Herpesvirus 6 Subtypes in Bone Marrow Transplant Patients by Using a Single Real-Time Polymerase Chain Reaction Assay

Human herpesvirus (HHV)-6 infections are ubiquitous, but infection or reactivation under immunocompromised conditions, such as bone marrow or solid organ transplantation, can often result in serious clinical manifestations. Two HHV-6 subtypes are known. Most primary HHV-6 infections are caused by subtype 6B, but little information is available about the prevalence, distribution, and clinical divergence of 6A and 6B. To study this, we have developed a highly sensitive and specific real-time polymerase chain reaction (PCR) assay that can detect, quantitate, and reliably differentiate HHV-6A and -6B in clinical specimens. Exploiting a single-base variation in the DNA polymerase gene of these respective subtypes, we used melting curve analysis for subtype discrimination. Moreover, this assay’s ability to discriminate HHV-6 subtypes was confirmed by PCR/restriction fragment length polymorphism analysis of the HHV-6 large tegument protein gene and PCR amplicon size-discrimination analysis of the HHV-6 immediate-early gene. Using this assay, we present our findings about the prevalence and distribution of these subtypes in bone marrow transplant patients. Of 803 plasma specimens tested from 353 patients, 136 specimens (17%) from 60 patients were determined to be HHV-6 positive. We analyzed these HHV-6-positive patients for subtype identification by using our newly developed assay and determined that 58 patients (97%) were HHV-6B positive and 2 patients (3%) were HHV-6A positive. No patient was coinfected with both subtypes. This assay can be a sensitive, genotype-specific, rapid method to reliably diagnose life-threatening HHV-6 infections in immunocompromised patients and can be useful in guiding and monitoring specific therapy.

Genotypic characterization of Burkholderia cenocepacia strains by rep-PCR and PCR–RFLP of the fliC gene

Thirty-five strains of Burkholderia cenocepacia from clinical and environmental sources were characterized genotypically by repetitive sequence PCR (ERIC- and BOX-PCR) and polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis of the flagellin gene ( fliC). In cluster analysis based on the repetitive PCR profiles the strains were composed of five clusters, of which clusters 1, 2 and 3 were more closely related to each other than to clusters 4 and 5. It has been reported that the majority of Burkholderia cepacia complex strains can be separated into two types on the basis of fliC size (types I and II correspond to 1.4 and 1.0 kb, respectively). When the strains were analysed by PCR of fliC, all strains yielded amplified products of 1.0 kb except for three strains. The latter strains gave PCR products of 0.7 kb (atypical type), which belonged to repetitive PCR cluster 5. These results indicated that the majority of B. cenocepacia strains belonged to flagellin type II. In the RFLP analysis of the large fliC amplicons with HaeIII, 10 patterns were observed indicating remarkable variation. Strains grouping in repetitive PCR cluster 4 had a unique fliC RFLP pattern. The results of repetitive PCR typing and PCR–RFLP analysis of fliC showed a strong correlation. Strains belonging to the repetitive PCR clusters 4 or 5 were distinctly different from other B. cenocepacia strains as shown by PCR–RFLP analysis of the fliC gene and phenotypic assays.

Optimization of the PAXgene™ blood RNA extraction system for gene expression analysis of clinical samples

One major problem associated with collecting whole blood from patients for use as a source of RNA in gene expression studies is that the RNA degrades during collection and storage. Preservation of RNA quality is vital in such studies because the stability of the RNA ultimately affects analysis of gene expression. In this study the PAXgene blood collection system was compared with a standard erythrocyte lysis method for isolating RNA from blood samples. The methods were compared in terms of RNA yield, RNA stabilization, and DNA contamination. The study also included the downstream application to RT-PCR analysis for relative mRNA expression levels of the ribonucleotide reductase subunits R1 and R2. The results show that blood collection in conventional collection tubes, and leukocyte isolation by erythrocyte lysis lead to significant degradation of RNA. Our findings confirm the ability of PAXgene to stabilize RNA in whole blood; however, RNA extracted by the PAXgene method contained significant DNA contamination. Given the low basal expression of the target genes analyzed in this study, contaminating DNA could potentially affect accurate interpretation of RT-PCR data. As a result, the PAXgene protocol was optimized to include off-column DNase treatments, which yielded high-quality RNA suitable for gene expression studies. Furthermore, the results suggest that RNA isolation with PAXgene is advantageous compared to traditional extraction methods for RT-PCR analysis of large or different-sized amplicons.

Molecular characterization of nuclear small subunit (18S)-rDNA pseudogenes in a symbiotic dinoflagellate (Symbiodinium, Dinophyta).

For the dinoflagellates, an important group of single-cell protists, some nuclear rDNA phylogenetic studies have reported the discovery of rDNA pseudogenes. However, it is unknown if these aberrant molecules are confined to free-living taxa or occur in other members of the group. We have cultured a strain of symbiotic dinoflagellate, belonging to the genus Symbiodinium, which produces three distinct amplicons following PCR for nuclear small subunit (18S) rDNA genes. These amplicons contribute to a unique restriction fragment length polymorphism pattern diagnostic for this particular strain. Sequence analyses revealed that the largest amplicon was the expected region of 18S-rDNA, while the two smaller amplicons are Symbiodinium nuclear 18S-rDNA genes that contain single long tracts of nucleotide deletions. Reverse transcription (RT)-PCR experiments did not detect RNA transcripts of these latter genes, suggesting that these molecules represent the first report of nuclear 18S-rDNA pseudogenes from the genome of Symbiodinium. As in the free-living dinoflagellates, nuclear rDNA pseudogenes are effective indicators of unique Symbiodinium strains. Furthermore, the evolutionary pattern of dinoflagellate nuclear rDNA pseudogenes appears to be unique among organisms studied to date, and future studies of these unusual molecules will provide insight on the cellular biology and genomic evolution of these protists.

Identifying and validating causal genetic alterations in human breast cancer.

An important mechanism for the activation of proto-oncogenes in human breast and other cancers is gene amplification, which results in gene overexpression at both the message and the protein levels. Recent studies have demonstrated that oncogenes rarely if ever become amplified in isolation, but rather are present on large amplicons that contain multiple genes. More detailed analysis of these amplicons has revealed the presence of many candidate breast cancer oncogenes. The broad goal of this issue of Breast Cancer Research and Treatment is to review the current state of our understanding of the causal role of defined genetic alterations that occur in human breast cancers, and to discuss the case for the mechanistic significance of several candidate oncogenes. As will be seen, these studies have revealed a remarkable genetic complexity and heterogeneity in human breast cancer that must be dissected in order to improve our mechanistic understanding of disease progression, and to develop effective new drugs against relevant molecular targets.

Variant Alleles on the Penta E Locus in the PowerPlex® 16 Kit

Penta E in the PowerPlex 16 kit is a pentanucleotide tandem repeat marker located on Chromosome 15, containing an AAAGA repeat motif. Variant alleles (18.4 and 19.4) were found in the Japanese population. A sequence analysis revealed that both the variant alleles had a partial repeat motif of AAAA, resulting in one-base-shorter alleles compared to known alleles. Despite the relatively large amplicon sizes (379 to 474 bp) of Penta E, an accurate allele assignment can be reliably made by capillary electrophoresis. However, alleles differing in size by only one base (e.g., 18.4 and 19) were not separated and appeared as a single broad peak. The Genotyper software assigned one of the component alleles to this peak. Therefore, such broad peaks require careful interpretation so as to not overlook the other component allele contained by the peak. As an index to recognize a peak containing two alleles, the ratio of peak area to peak height was found to be useful.

Chromosomal Localization of DNA Amplifications in Neuroblastoma Tumors Using cDNA Microarray Comparative Genomic Hybridization

Conventional comparative genomic hybridization (CGH) profiling of neuroblastomas has identified many genomic aberrations, although the limited resolution has precluded a precise localization of sequences of interest within amplicons. To map high copy number genomic gains in clinically matched stage IV neuroblastomas, CGH analysis using a 19,200-feature cDNA microarray was used. A dedicated (freely available) algorithm was developed for rapid in silico determination of chromosomal localizations of microarray cDNA targets, and for generation of an ideogram-type profile of copy number changes. Using these methodologies, novel gene amplifications undetectable by chromosome CGH were identified, and larger MYCN amplicon sizes (in one tumor up to 6 Mb) than those previously reported in neuroblastoma were identified. The genes HPCAL1, LPIN1/KIAA0188, NAG, and NSE1/LOC151354 were found to be coamplified with MYCN. To determine whether stage IV primary tumors could be further subclassified based on their genomic copy number profiles, hierarchical clustering was performed. Cluster analysis of microarray CGH data identified three groups: 1) no amplifications evident, 2) a small MYCN amplicon as the only detectable imbalance, and 3) a large MYCN amplicon with additional gene amplifications. Application of CGH to cDNA microarray targets will help to determine both the variation of amplicon size and help better define amplification-dependent and independent pathways of progression in neuroblastoma.

Human cytomegalovirus DNA in plasma and serum specimens of renal transplant recipients is highly fragmented.

Quantitation of cytomegalovirus (CMV) DNA in plasma and serum by PCR is increasingly used to identify patients at risk for developing CMV disease and to monitor the efficacy of antiviral therapy. Although CMV DNA levels are generally interpreted as viral loads, the exact nature of the viral DNA in these specimens is unknown. We studied the state of CMV DNA in plasma and serum specimens obtained from three renal transplant recipients at peak viral DNA levels during primary CMV infection. For this purpose, DNA isolated from these specimens was fractionated by size, and CMV DNA levels in the resulting DNA fractions were measured by quantitative PCR targeted at large (578-bp) and small (134-bp) amplicons. These experiments showed that the molecular sizes of DNA fragments from which CMV DNA is amplified were small (<2,000 bp), indicating that CMV DNA in plasma and serum is highly fragmented. Furthermore, CMV DNA levels were consistently higher when targeted at the smaller amplicon, providing additional evidence for the fragmentation of viral DNA. In conclusion, the first results with three patients have shown that CMV DNA in plasma and serum is highly fragmented and does not necessarily reflect the amount of infectious virus. These observations have potential consequences for understanding CMV pathogenesis and interpreting CMV DNA levels in individual patient management.

Polymerase chain reaction for detection of avian leukosis virus subgroup J in feather pulp.

Feather pulp from experimentally infected chickens was used as a source of DNA for polymerase chain reaction (PCR) amplification of avian leukosis virus subgroup J (ALV-J) proviral DNA. A primer set that produces a large amplicon (approximately 2,125) was used to detect ALV-J proviral DNA. This primer set was used in lieu of previously published primers because it allows for sequencing of the entire envelope gene and because it was able to detect diagnostically a number of North American ALV-J isolates that could not be detected with previously published primers and PCR conditions. ALV-J proviral DNA was detected in feather pulp at 7 days of age in more than 90% of birds infected as embryos and 7 days postinoculation in over 50% of chickens infected at 3 days of age. The results obtained with PCR on feather pulp were compared with those of virus isolation. In the embryo-inoculated birds, the percentages of agreement between PCR and virus isolation were 92.5% at 7 days of age and 100% at 28, 42, 49, and 56 days of age. However, the overall sensitivity of virus isolation in embryo-infected birds was higher, particularly at 7 and 56 days of age. In chickens inoculated at 3 days of age, the percentages of agreement of detection between PCR and virus isolation ranged from 75% at 10 days of age to 100% at 42 days of age. Agreement of negative results of ALV-J detection by PCR and virus isolation in chickens infected posthatch ranged between 66.6% and 100% between the ages of 10 and 42 days. Virus isolation requires chicken embryo fibroblasts of specific genetic lines, and the process takes onaverage 7-9 days. Aseptic collection of blood and tissues for virus isolation and molecular detection of ALV-J requires sterile necropsy instruments as well as syringes and needles for each individual chicken, whereas sterile microcentrifuge tubes and gloves are the only equipment necessary for aseptic feather pulp collection for ALV-J detection by PCR. PCR-based detection of ALV-J in feather pulp is especially suitable when ALV-J infection must be diagnosed rapidly and unequivocally without killing the chicken(s) and in situations where crucial reagents or suitable virus propagation substrates are not readily available for isolation and propagation of ALV-J in cell culture.

Detection of double-stranded PCR amplicons at the attomole level electrosprayed from low nanomolar solutions using FT-ICR mass spectrometry.

An 82-base-pair polymerase chain reaction (PCR) product was amplified from the tetranucleotide short tandem repeat locus within the human tyrosine hydroxylase gene. PCR amplification was carried out using 100 ng of human nuclear DNA obtained from an individual who is homozygotic for the 9.3 allele resulting in a 50.5 kDa amplicon. To generate sufficient material for these investigations, several reactions were pooled and subsequently purified and quantified using UV-vis spectrophotometry. A serial dilution was carried out from a 2 microM stock solution providing solution concentrations down to 5 nM. Measurements were made using hexapole accumulation and gated trapping strategies in a 4.7 Telsa Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS) which facilitated detection of the amplicon at the attomole level when electrosprayed from a 5 nM solution with a single acquisition! The signal-to-noise ratio was determined to be 8.3 for the spectrum derived from the 5 nM solution using the magnitude-mode mass spectral peak height for the most abundant charge-state. This remarkable sensitivity for large PCR amplicons will dramatically improve the ability of electrospray ionization mass spectrometry to address important genetic questions for low copy number genes or when the amount of initial template is limited; the latter issue is commonly encountered in DNA forensics. Furthermore, these data represents over 2 orders of magnitude decrease in detection limits over other existing ESI-MS reports concerning PCR products, including those conducted using FTICR-MS.