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Selective recovery of founder genetic diversity in aquacultural broodstocks and captive, endangered fish populations.

Hatchery broodstocks used for genetic conservation or aquaculture may represent their ancestral gene pools rather poorly. This is especially likely when the fish that found a broodstock are close relatives of each other. We re-analysed microsatellite data from a breeding experiment on red sea bream to demonstrate how lost genetic variation might be recovered when gene frequencies have been distorted by consanguineous founders in a hatchery. A minimal-kinship criterion based on a relatedness estimator was used to select subsets of breeders which represented the maximum number of founder lineages (i.e., carried the fewest identical copies of ancestral genes). UPGMA clustering of Nei's genetic distances grouped these selected subsets with the parental gene pool, rather than with the entire, highly 'drifted' offspring generation. The selected subsets also captured much of the expected heterozygosity and allelic diversity of the parental gene pool. Independent pedigree data on the same fish showed that the selected subsets had more contributing parents and more founder equivalents than random subsets of the same size. The estimated mean coancestry was lower in the selected subsets, meaning that inbreeding in subsequent generations would be lower if they were used as breeders. The procedure appears suitable for reducing the genetic distortion due to consanguineous and over-represented founders of a hatchery gene pool.

Alleles↗

Large-scale structure of genomic methylation patterns.

The mammalian genome depends on patterns of methylated cytosines for normal function, but the relationship between genomic methylation patterns and the underlying sequence is unclear. We have characterized the methylation landscape of the human genome by global analysis of patterns of CpG depletion and by direct sequencing of 3073 unmethylated domains and 2565 methylated domains from human brain DNA. The genome was found to consist of short (<4 kb) unmethylated domains embedded in a matrix of long methylated domains. Unmethylated domains were enriched in promoters, CpG islands, and first exons, while methylated domains comprised interspersed and tandem-repeated sequences, exons other than first exons, and non-annotated single-copy sequences that are depleted in the CpG dinucleotide. The enrichment of regulatory sequences in the relatively small unmethylated compartment suggests that cytosine methylation constrains the effective size of the genome through the selective exposure of regulatory sequences. This buffers regulatory networks against changes in total genome size and provides an explanation for the C value paradox, which concerns the wide variations in genome size that scale independently of gene number. This suggestion is compatible with the finding that cytosine methylation is universal among large-genome eukaryotes, while many eukaryotes with genome sizes <5 x 10(8) bp do not methylate their DNA.

Animals↗

Quantitation of blood lymphocyte mitochondrial DNA for the monitoring of antiretroviral drug-induced mitochondrial DNA depletion.

OBJECTIVE: To investigate the impact of antiretroviral treatment on the mitochondrial DNA (mtDNA) content of peripheral blood mononuclear cells (PBMCs) from HIV-1-infected patients. DESIGN: As absolute mtDNA copy numbers widely differ between individuals, we performed a longitudinal analysis where the patient's first historical specimen was obtained as a baseline reference for relative comparison with subsequent samples from that patient. METHODS: mtDNA and nuclear DNA quantitation per cell (beta-globin gene copies) were both measured by real-time polymerase chain reaction analysis of whole DNA extracts of 361 serial live-cryopreserved PBMCs collected in former trials and clinical follow-ups from 60 individuals with established or recently acquired HIV-1 infections before and during administration of various antiviral combination therapies. RESULTS: mtDNA amounts were stable or increasing over years of natural HIV-1 infection in untreated patients (n = 7), consistent with our finding of a lack of differences in mtDNA copy numbers in patients with either a long established or recent lentivirus infection. Our quantitation system revealed significant changes in mtDNA copy number depending on the designated triple, quadruple, or quintuple anti-HIV drug combinations. Zidovudine + zalcitabine + ritonavir and zidovudine + lamivudine + didanosine regularly lead to mtDNA depletion in each of the treated patients, whereas none of 7 patients (and 35 cell specimens) receiving a stavudine + lamivudine + indinavir combination had any significant mtDNA content variations. In 7 patients, mtDNA copy numbers returned to pretreatment levels and/or higher levels without any interruption of the previously mtDNA-depleting antiretroviral drug combination. CONCLUSION: Our assay system allowed the detection of significant changes in the mtDNA content of PBMCs from HIV-1-infected patients taking antiretroviral drugs, as has been reported in the literature with other detection systems. Yet, mtDNA copy numbers regularly diminished during administration of some but not all nucleoside analog-containing combinations. This, plus the occasional finding that depleted mtDNA contents spontaneously increased to baseline levels and/or higher levels during uninterrupted treatment, should raise a note of caution about resorting to the PBMC mtDNA marker for monitoring of antiretroviral drug-related mitochondrial toxicities.

Acquired Immunodeficiency Syndrome↗

Domain swapping and gene shuffling identify sequences required for induction of an Avr-dependent hypersensitive response by the tomato Cf-4 and Cf-9 proteins.

The tomato Cf-4 and Cf-9 genes confer resistance to infection by the biotrophic leaf mold pathogen Cladosporium. Their protein products induce a hypersensitive response (HR) upon recognition of the fungus-encoded Avr4 and Avr9 peptides. Cf-4 and Cf-9 share >91% sequence identity and are distinguished by sequences in their N-terminal domains A and B, their N-terminal leucine-rich repeats (LRRs) in domain C1, and their LRR copy number (25 and 27 LRRs, respectively). Analysis of Cf-4/Cf-9 chimeras, using several different bioassays, has identified sequences in Cf-4 and Cf-9 that are required for the Avr-dependent HR in tobacco and tomato. A 10-amino acid deletion within Cf-4 domain B relative to Cf-9 was required for full Avr4-dependent induction of an HR in most chimeras analyzed. Additional sequences required for Cf-4 function are located in LRRs 11 and 12, a region that contains only eight of the 67 amino acids that distinguish it from Cf-9. One chimera, with 25 LRRs that retained LRR 11 of Cf-4, induced an attenuated Avr4-dependent HR. The substitution of Cf-9 N-terminal LRRs 1 to 9 with the corresponding sequences from Cf-4 resulted in attenuation of the Avr9-induced HR, as did substitution of amino acid A433 in LRR 15. The amino acids L457 and K511 in Cf-9 LRRs 16 and 18 are essential for induction of the Avr9-dependent HR. Therefore, important sequence determinants of Cf-9 function are located in LRRs 10 to 18. This region contains 15 of the 67 amino acids that distinguish it from Cf-4, in addition to two extra LRRs. Our results demonstrate that sequence variation within the central LRRs of domain C1 and variation in LRR copy number in Cf-4 and Cf-9 play a major role in determining recognition specificity in these proteins.

Amino Acid Sequence↗

Physical and genetic analysis of IS110, a transposable element of Streptomyces coelicolor A3(2).

On at least three independent occasions a 1.6 kb segment of Streptomyces coelicolor DNA was detected in apparently the same location in an attP-deleted derivative of the temperate phage phiC31 that carried a selectable viomycin resistance gene. This sequence (termed IS110) allowed integration of the phage (giving viomycin-resistant transductants) at homologous sequences (detected by Southern hybridisation) at several locations in the S. coelicolor genome. The inserted prophages facilitated genetic mapping of two IS110 copies in the chromosomal linkage map. A third copy did not exhibit simple segregation with chromosomal markers, and there appeared to be a frequent DNA rearrangement close to this copy. Some variation in the number of copies of IS110 and their location has taken place in the pedigree of S. coelicolor derivatives. IS110 did not hybridise to any known S. coelicolor plasmid, nor to any of several other IS-like elements previously described in other Streptomyces plasmids or phages. It hybridised strongly to DNA from only a small minority of other Streptomyces species and was absent from S. lividans, a close relative of S. coelicolor.

Bacteriophages↗

Genome-wide analysis of C/D and H/ACA-like small nucleolar RNAs in Leishmania major indicates conservation among trypanosomatids in the repertoire and in their rRNA targets.

Small nucleolar RNAs (snoRNAs) are a large group of noncoding RNAs that exist in eukaryotes and archaea and guide modifications such as 2'-O-ribose methylations and pseudouridylation on rRNAs and snRNAs. Recently, we described a genome-wide screening approach with Trypanosoma brucei that revealed over 90 guide RNAs. In this study, we extended this approach to analyze the repertoire of the closely related human pathogen Leishmania major. We describe 23 clusters that encode 62 C/Ds that can potentially guide 79 methylations and 37 H/ACA-like RNAs that can potentially guide 30 pseudouridylation reactions. Like T. brucei, Leishmania also contains many modifications and guide RNAs relative to its genome size. This study describes 10 H/ACAs and 14 C/Ds that were not found in T. brucei. Mapping of 2'-O-methylations in rRNA regions rich in modifications suggests the existence of trypanosomatid-specific modifications conserved in T. brucei and Leishmania. Structural features of C/D snoRNAs, such as copy number, conservation of boxes, K turns, and intragenic and extragenic base pairing, were examined to elucidate the great variation in snoRNA abundance. This study highlights the power of comparative genomics for determining conserved features of noncoding RNAs.

Animals↗

Genotypic variation and stability of four variable-number tandem repeats and their suitability for discriminating strains of Mycobacterium leprae.

It has not been possible to distinguish different strains of Mycobacterium leprae according to their genetic sequence. However, the genome contains several variable-number tandem repeats (VNTR), which have been used effectively in strain typing of other bacteria. To determine their suitability for differentiating M. leprae, we developed PCR systems to amplify 5 different VNTR loci and examined a battery of 12 M. leprae strains derived from patients in different regions of the United States, Brazil, Mexico, and the Philippines, as well as from wild armadillos and a sooty mangabey monkey. We found diversity at four VNTR (D = 0.74), but one system (C(16)G(8)) failed to yield reproducible results. Alleles for the GAA VNTR varied in length from 10 to 16 copies, those for AT(17) varied in length from 10 to 15 copies, those for GTA varied in length from 9 to 12 copies, and those for TA(18) varied in length from 13 to 20 copies. Relatively little variation was seen with interspecies transfer of bacilli or during short-term passage of strains in nude mice or armadillos. The TA(18) locus was more polymorphic than other VNTR, and genotypic variation was more common after long-term expansion in armadillos. Most strain genotypes remained fairly stable in passage, but strain Thai-53 showed remarkable variability. Statistical cluster analysis segregated strains and passage samples appropriately but did not reveal any particular genotype associable with different regions or hosts of origin. VNTR polymorphisms can be used effectively to discriminate M. leprae strains. Inclusion of additional loci and other elements will likely lead to a robust typing system that can be used in community-based epidemiological studies and select clinical applications.

Genetic Variation↗

Species and recombination effects on DNA variability in the tomato genus.

Population genetics theory predicts that strong selection for rare, beneficial mutations or against frequent, deleterious mutations reduces polymorphism at linked neutral (or weakly selected) sites. The reduction of genetic variation is expected to be more severe when recombination rates are lower. In outbreeding species, low recombination rates are usually confined to certain chromosomal regions, such as centromeres and telomeres. In contrast, in predominantly selfing species, the rarity of double heterozygotes leads to a reduced effective recombination rate in the whole genome. We investigated the effects of restricted recombination on DNA polymorphism in these two cases, analyzing five Lycopersicon species with contrasting mating systems: L. chilense, L. hirsutum, L. peruvianum, L. chmielewskii, and L. pimpinellifolium, of which only the first three species have self-incompatibility alleles. In each species, we determined DNA sequence variation of five single-copy genes located in chromosomal regions with either high or low recombination rate. We found that the mating system has a highly significant effect on the level of polymorphism, whereas recombination has only a weak influence. The effect of recombination on levels of polymorphism in Lycopersicon is much weaker than in other well-studied species, including Drosophila. To explain these observations, we discuss a number of hypotheses, invoking selection, recombination, and demographic factors associated with the mating system. We also provide evidence that L. peruvianum, showing a level of polymorphism (almost 3%) that is comparable to the level of divergence in the whole genus, is the ancestral species from which the other species of the genus Lycopersicon have originated relatively recently.

DNA↗

Identification of genetic aberrations on chromosome 22 outside the NF2 locus in schwannomatosis and neurofibromatosis type 2.

Schwannomatosis is characterized by multiple peripheral and cranial nerve schwannomas that occur in the absence of bilateral 8th cranial nerve schwannomas. The latter is the main diagnostic criterion of neurofibromatosis type 2 (NF2), which is a related but distinct disorder. The genetic factors underlying the differences between schwannomatosis and NF2 are poorly understood, although available evidence implicates chromosome 22 as the primary location of the gene(s) of interest. To investigate this, we comprehensively profiled the DNA copy number in samples from sporadic and familial schwannomatosis, NF2, and a large cohort of normal controls. Using a tiling-path chromosome 22 genomic array, we identified two candidate regions of copy number variation, which were further characterized by a PCR-based array with higher resolution. The latter approach allows the detection of minute alterations in total genomic DNA, with as little as 1.5 kb per measurement point of nonredundant sequence on the array. In DNA derived from peripheral blood from a schwannomatosis patient and a sporadic schwannoma sample, we detected rearrangements of the immunoglobulin lambda (IGL) locus, which is unlikely to be due to a B-cell specific somatic recombination of IGL. Analysis of normal controls indicated that these IGL rearrangements were restricted to schwannomatosis/schwannoma samples. In the second candidate region spanning GSTT1 and CABIN1 genes, we observed a frequent copy number polymorphism at the GSTT1 locus. We further describe missense mutations in the CABIN1 gene that are specific to samples from schwannomatosis and NF2 and make this gene a plausible candidate for contributing to the pathogenesis of these disorders.

Adaptor Proteins, Signal Transducing↗

Prenatal diagnosis of chromosomal abnormalities using array-based comparative genomic hybridization.

PURPOSE: This study was designed to evaluate the feasibility of using a targeted array-CGH strategy for prenatal diagnosis of genomic imbalances in a clinical setting of current pregnancies. METHODS: Women undergoing prenatal diagnosis were counseled and offered array-CGH (BCM V4.0) in addition to routine chromosome analysis. Array-CGH was performed with DNA directly from amniotic fluid cells with whole genome amplification, on chorionic villus samples with amplification as necessary, and on cultured cells without amplification. RESULTS: Ninety-eight pregnancies (56 amniotic fluid and 42 CVS specimens) were studied with complete concordance between karyotype and array results, including 5 positive cases with chromosomal abnormalities. There was complete concordance of array results for direct and cultured cell analysis in 57 cases tested by both methods. In 12 cases, the array detected copy number variation requiring testing of parental samples for optimal interpretation. Array-CGH results were available in an average of 6 and 16 days for direct and cultured cells, respectively. Patient acceptance of array-CGH testing was 74%. CONCLUSION: This study demonstrates the feasibility of using array-CGH for prenatal diagnosis, including reliance on direct analysis without culturing cells. Use of array-CGH should increase the detection of abnormalities relative to the risk, and is an option for an enhanced level of screening for chromosomal abnormalities in high risk pregnancies.

Chromosome Aberrations↗

Quantification of G protein mRNA using reverse transcription and competitive PCR with a colorimetric microplate assay.

This paper reports an assay for the quantification of levels of specific mRNA for the alpha subunits of the inhibitory G proteins G alpha i-1, G alpha i-2, and G alpha i-3. The assay employs reverse transcription and competitive polymerase chain reaction (PCR) coupled to enzyme-linked oligonucleotide sorbent assay for differential detection and quantification of PCR products. The assay was conducted with conventional thermal block PCR cyclers as well as rapid air microcapillary cyclers. The detection stage consists of three steps using synthetic oligonucleotides, commercially available reagents and a conventional 96-well plate absorbance reader at settings of 450 and 630 nm. The assay is: (1) rapid, requiring about 3 h for quantification of PCR products; (2) safe, being non-radiometric; (3) relatively simple; (4) highly sensitive, being capable of detecting less than 10 initial copies of target cDNA; (5) precise, resolving two-fold differences in initial copy numbers of specific sequences as low as 10(-20) mol; (6) linear over a 3 log range, with two-fold differences in the quantity of cDNA producing consistent reductions in quantity of specific cDNA detected; and (7) reproducible, intra-assay and inter-assay coefficients of variation being 11.9 and 14.7%, respectively.

Animals↗

Effect of dietary cholesterol on low density lipoprotein-receptor, 3-hydroxy-3-methylglutaryl-CoA reductase, and low density lipoprotein receptor-related protein mRNA expression in healthy humans.

We investigated the possibility that dietary cholesterol downregulates the expression of low density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase genes of circulating mononuclear cells in vivo in healthy humans. We also studied the variations of the LDL receptor-related protein (LRP) gene in the same conditions. Dieters (n = 5) were submitted to a 4-d fat restriction (mean cholesterol intake: 6+/-4 mg/d), followed by a 7-d cholesterol (a mean of 791+/-150 mg/d) supplementation. Controls (n = 3) did not change their diet. During fat restriction, serum total and LDL cholesterol decreased significantly (P < 0.05), and LDL receptor and HMG-CoA reductase mRNA copy numbers in mononuclear cells increased by 57 and 147%, respectively (P < 0.05). After reintroducing cholesterol, serum cholesterol was stable whereas LDL receptor and HMG-CoA reductase mRNA decreased by 46 and 72% (P < 0.05) and LRP mRNA increased by 59% (P < 0.005). The changes in LDL receptor and HMG-CoA reductase mRNA abundance were correlated (r = +0.79, P = 0.02) during cholesterol reintroduction as were LDL receptor and LRP mRNA levels, but negatively (r = -0.70, P = 0.05). Also, 70% of the variability in LRP mRNA (P < 0.005) was explained by dietary cholesterol. Thus, the basic mechanisms regulating cellular cholesterol content, the coordinate feedback repression of genes governing the synthesis and uptake of cholesterol, are operating in vivo in humans. However, serum cholesterol did not increase in response to dietary cholesterol, suggesting that these mechanisms may not play as predominant a role as previously believed in the short-term control of serum cholesterol in vivo in humans. A new finding is that LRP gene is also sensitive to dietary cholesterol, suggesting that it may participate in the control of serum cholesterol. Further in vivo studies in humans are warranted to explore the molecular mechanisms of the physiological response to dietary cholesterol in humans.

Adult↗

Contour area filtering of two-dimensional electrophoresis images.

We describe an algorithm, Contour Area Filtering, for separating background from foreground in gray scale images. The algorithm is based on the area contained within gray scale contour lines. It can be viewed as a form of local thresholding, or as a seed growing algorithm, or as a type of watershed segmentation. The most important feature of the algorithm is that it uses object area to determine the segmentation. Thus, it is relatively impervious to brightness and contrast variations across an image or between different images. Contour Area Filtering was designed specifically for image analysis of 2D electrophoresis gels, although it can be applied to other gray scale images. A typical gel image is an electrophoretogram or a phosphor image of 1000-2500 spots representing protein or DNA restriction fragments. The images are quantitative with spot intensities reflective of the number of proteins or the DNA fragment copy number. The background intensity can vary widely across the image caused both by variation in spot density and by the physical laboratory process of creating a gel. Analyzing and comparing gel images entails extracting and segmenting spots, registering images and matching spots, and measuring differences between spots. We present experimental results which show that Contour Area Filtering is a quick, efficient method for separating electrophoresis gel background from foreground with extremely high accuracy.

Algorithms↗

Different evolutionary behaviour of P element subfamilies: M-type and O-type elements in Drosophila bifasciata and D. imaii.

Distribution and variation of two P-element subfamilies designated M-type and O-type elements were investigated in Drosophila bifasciata (Db) and its relatives. PCR screening revealed that full-sized and internally deleted elements of both types occur in three geographic Db strains and in the related species, D. imaii (Di). Molecular analyses indicate differences in the evolutionary behaviour of the two P-element types. Internally deleted M-type elements fall into two size classes present in all three Db strains. In contrast, internally deleted O-type elements vary between the strains in number and length. With respect to genomic location, M-type elements seem to be restricted to conserved euchromatic sites, whereas the positions of O-type elements appear to be geographically variable. In one strain of Db (Italy), O-type elements seem to accumulate in the heterochromatin. Sequencing of a 397-bp segment shows intra- and interspecific divergence of M-type elements. In a 452-bp segment of the O-type elements, no substitutions were found, neither within nor between species. This finding suggests recent introgression of O-type elements via hybridization between Db and Di. Sequence identity and variation in chromosomal locations among different copies imply that O-type elements are transpositionally active. For M-type elements, genomic mobility cannot be proved. In a survey of several other taxa, no O-type-related sequences were detected so far. Therefore, the origin of the O-type subfamily remains unknown, whereas the source of M-type elements can be traced back to the genus Scaptomyza.

Animals↗

Complex patterns of copy number variation at sites of segmental duplications: an important category of structural variation in the human genome.

The structural diversity of the human genome is much higher than previously assumed although its full extent remains unknown. To investigate the association between segmental duplications that display constitutive copy number differences (CNDs) between humans and the great apes and those which exhibit polymorphic copy number variations (CNVs) between humans, we analysed a BAC array enriched with segmental duplications displaying such CNDs. This study documents for the first time that in addition to human-specific gains common to all humans, these duplication clusters (DCs) also exhibit polymorphic CNVs > 40 kb. Segmental duplication is known to have been a frequent event during human genome evolution. Importantly, among the CNV-associated genes identified here, those involved in transcriptional regulation were found to be significantly overrepresented. Complex patterns of variation were evident at sites of DCs, manifesting as inter-individual differentially sized copy number alterations at the same genomic loci. Thus, CNVs associated with segmental duplications do not simply represent insertion/deletion polymorphisms, but rather constitute a wide variety of rearrangements involving differential amplification and partial gains and losses with high inter-individual variability. Although the number of CNVs was not found to differ between Africans and Caucasians/Asians, the average number of variant patterns per locus was significantly lower in Africans. Thus, complex variation patterns characterizing segmental duplications result from relatively recent genomic rearrangements. The high number of these rearrangements, some of which are potentially recurrent, together with differences in population size and expansion dynamics, may account for the greater diversity of CNV in Caucasians/Asians as compared with Africans.

Animals↗

Integrated single-cell and spatial transcriptomic analyses reveal malignant epithelial glycolytic heterogeneity and spatial niche remodeling during colorectal cancer progression.

Colorectal cancer (CRC) progression is shaped by metabolic reprogramming and complex interactions within the tumor microenvironment. However, the cellular heterogeneity, spatial organization, and clinical relevance of glycolytic activity in CRC remain incompletely understood. In this study, we integrated single-cell RNA sequencing, bulk transcriptomics, and spatial transcriptomics data to systematically characterize glycolytic heterogeneity in CRC. Glycolytic activity was quantified using five independent scoring methods, consistently showing that epithelial cells exhibited the highest glycolytic activity across the two single-cell cohorts. Stratification of CopyKAT-verified aneuploid malignant epithelial cells into high-glycolysis (HG) and low-glycolysis (LG) subgroups by glycolysis scores revealed that HG cells exhibited higher stemness scores and chromosomal copy number variations. Cell-cell communication analysis revealed that, compared with LG cells, HG cells exhibited increased interaction frequency and strength with immune and stromal populations, indicating enhanced malignant epithelial-microenvironment crosstalk. Spatial transcriptomics analyses further revealed that glycolytic activity varied across normal colorectal tissue, primary CRC, and colorectal liver metastases, accompanied by progressive remodeling of epithelial-associated spatial niches and MIF-mediated intercellular communication. Bulk transcriptomic analysis identified a glycolysis-related prognostic signature with robust predictive performance, which served as an independent prognostic factor for overall survival in CRC cohorts. Collectively, these findings indicate that glycolytic heterogeneity is a key feature of CRC malignant epithelial cells and is closely associated with tumor progression, microenvironmental remodeling, and clinical outcomes.

Humans↗

Periodic extinctions of transposable elements in bacterial lineages: evidence from intragenomic variation in multiple genomes.

Most previous work on the evolution of mobile DNA was limited by incomplete sequence information. Whole genome sequences allow us to overcome this limitation. I study the nucleotide diversity of prominent members of five insertion sequence families whose transposition activity is encoded by a single transposase gene. Eighteen among 376 completely sequenced bacterial genomes and plasmids carry between 3 and 20 copies of a given insertion sequence. I show that these copies generally show very low DNA divergence. Specifically, more than 68% of the transposase genes are identical within a genome. The average number of amino acid replacement substitutions at amino acid replacement sites is Ka = 0.013, that at silent sites is Ks = 0.1. This low intragenomic diversity stands in stark contrast to a much higher divergence of the same insertion sequences among distantly related genomes. Gene conversion among protein-coding genes is unlikely to account for this lack of diversity. The relation between transposition frequencies and silent substitution rates suggests that most insertion sequences in a typical genome are evolutionarily young and have been recently acquired. They may undergo periodic extinction in bacterial lineages. By implication, they are detrimental to their host in the long run. This is also suggested by the highly skewed and patchy distribution of insertion sequences among genomes. In sum, one can think of insertion sequences as slow-acting infectious diseases of cell lineages.

Bacteria↗

Rapid quantification of hepatitis B virus DNA by real-time PCR using efficient TaqMan probe and extraction of virus DNA.

AIM: To rapidly quantify hepatitis B virus (HBV) DNA by real-time PCR using efficient TaqMan probe and extraction methods of virus DNA. METHODS: Three standards were prepared by cloning PCR products which targeted S, C and X region of HBV genome into pGEM-T vector respectively. A pair of primers and matched TaqMan probe were selected by comparing the copy number and the Ct values of HBV serum samples derived from the three different standard curves using certain serum DNA. Then the efficiency of six HBV DNA extraction methods including guanidinium isothiocyanate, proteinase K, NaI, NaOH lysis, alkaline lysis and simple boiling was analyzed in sample A, B and C by real-time PCR. Meanwhile, 8 clinical HBV serum samples were quantified. RESULTS: The copy number of the same HBV serum sample originated from the standard curve of S, C and X regions was 5.7 multiply 10(4)/mL, 6.3 multiply 10(2)/mL and 1.6 multiply 10(3)/mL respectively. The relative Ct value was 26.6, 31.8 and 29.5 respectively. Therefore, primers and matched probe from S region were chosen for further optimization of six extraction methods. The copy number of HBV serum samples A, B and C was 3.49 multiply 10(9)/mL, 2.08 multiply 10(6)/mL and 4.40 multiply 10(7)/mL respectively, the relative Ct value was 19.9, 30 and 26.2 in the method of NaOH lysis, which was the efficientest among six methods. Simple boiling showed a slightly lower efficiency than NaOH lysis. Guanidinium isothiocyanate, proteinase K and NaI displayed that the copy number of HBV serum sample A, B and C was around 10(5)/mL, meanwhile the Ct value was about 30. Alkaline failed to quantify the copy number of three HBV serum samples. Standard deviation (SD) and coefficient variation (CV) were very low in all 8 clinical HBV serum samples, showing that quantification of HBV DNA in triplicate was reliable and accurate. CONCLUSION: Real-time PCR based on optimized primers and TaqMan probe from S region in combination with NaOH lysis is a simple, rapid and accurate method for quantification of HBV serum DNA.

Base Sequence↗