Detection and direct sequencing of hepatitis B virus genome by DNA amplification method

Hepatitis B virus (HBV) DNA was detected with amplification by the polymerase chain reaction method. Cloned HBV DNA equivalent to one virus genome (3 x 10(-6) pg) was detectable by ethidium bromide staining after 50 cycles of polymerase chain reaction. By applying this method, presence of HBV DNA was studied in 23 hepatitis B surface antigen (HBsAg)-positive and 11 HBsAg-negative sera from patients with chronic liver disease. Hepatitis B virus DNA was positive in 8 of 8 hepatitis B e antigen (HBeAg)-positive, in 2 of 2 HBeAg- and anti-HBe-negative, and in 12 of 13 anti-HBe-positive sera. Hepatitis B virus DNA was undetectable in all HBsAg-negative sera even with amplification. To confirm specificity, the amplified product was directly sequenced. Sequences around 122nd and 160th codon of HBs gene, which determines subtypes d/y and w/r, respectively, were analyzed. The results were compatible with recent reports regarding the relation between HBV subtypes and HBV DNA sequence at those codons. Hepatitis B virus DNA could be detected at the level of one virion by gene amplification method, and its sequence could be determined by direct sequencing in a few days.     

The pattern of mutational involvement of RAS genes in human hematologic malignancies determined by DNA amplification and direct sequencing

DNA from 161 patients with various forms of hematologic malignancies were investigated for mutations in exons 1 and 2 of the N-RAS, K-RAS and Ha-RAS gene by direct sequencing of DNA amplified in vitro by the polymerase chain reaction. Mutations involving either codons 11, 12, or 13 of the N-RAS gene were identified in 18 of the 161 patients. The relative frequencies of N-RAS gene mutations in these hematologic disorders was as follows: acute myelogenous leukemia (AML), 15%; acute lymphoblastic leukemia (ALL), 14%; myelodysplastic syndromes, 24%; and myeloid and lymphoid blast crisis of chronic myelogenous leukemia (CML), 3%. No correlation was observed between the presence of mutations and cytologic features or immunophenotype of these malignancies. Mutations involving codons 12 or 13 were equally prevalent, with a glycine to aspartic acid substitution being the most frequently encountered change. A single T-ALL case had a codon 11 mutation resulting in substitution of alanine with threonine. We failed to find mutations in exons 1 and 2 of the K-RAS or Ha-RAS genes in any case except a single AML with a mutation in codon 61 of the K-RAS gene. Also, no mutations were identified in chronic phase of CML, chronic lymphocytic leukemia. Ph1 positive ALL, non-Hodgkin's lymphoma, Hodgkin's disease, or multiple myeloma. These results indicate that RAS mutations, especially those involving exon 1 of the N-RAS gene, are frequent only in a subset of hematologic malignancies.     

Cloning and direct examination of a structurally abnormal human beta 0-thalassemia globin gene.

Restriction endonuclease mapping permitted identification of a form of beta 0-thalassemia in which a partial deletion of the beta-globin structural gene occurred [Orkin, S. H., Old, J. M., Weatherall, D. J. & Nathan, D. G. (1979) Proc. Natil. Acad. Sci. USA 76, 2400-2404]. To analyze its structure more directly, this abnormal human gene has now been cloned in bacteriophage lambda gtWES. Restriction mapping of the cloned gene and of a normal beta-globin gene contained in the phage H beta G1 confirmed previous findings regarding the presence of a deletion toward the 3' end of the gene but could not establish its position more accurately. Electron microscopy of the hybrid of the beta-thalassemia gene with globin RNA (R-loop analysis) provided unequivocal evidence for a deletion with the beta-globin structural gene. Hybridization of restriction fragments of the mutant gene with homologous fragments of H beta G1 (heteroduplex analysis) revealed a continuous, internal deletion of about 0.6 kilobase of DNA in the mutant gene and permitted its localization within the beta-globin gene region. This deletion removed the terminal third of the large intervening sequence within the beta-globin gene, the entire 3' coding block (extending from codon 105 to the end of the gene), and approximately 150 base pairs of DNA past the end of the normal globin gene.     

Detection of ras gene mutations of pancreatic tumors by polymerase chain reaction and direct sequencing method

Ras genes (H-,K-,N-ras) are converted to active oncogenes by point mutations occurring in either codon 12, 13 or 61. We analyzed 19 pancreatic tumors (formalin fixed paraffin embedded tissue) of these codons by a method to directly sequence nucleotides around codons 12/13 and 61 of the three ras genes, using polymerase chain reaction and direct sequencing method. Of 19 pancreatic tumors, all 17 duct cell carcinomas involving 2 mucous producing pancreatic cancers had point mutations of the K-ras codon 12, but 2 islet cell tumors had ano point mutation around codons 12, 13, 61 of the three ras genes. Extremely high incidence of ras gene mutation may be relevant to certain pathogenesis of pancreatic cancers.     

Analysis of N-RAS exon-1 mutations in myelodysplastic syndromes by polymerase chain reaction and direct sequencing

Mutations in codons 12 or 13 of the first exon of the N-RAS gene have been reported in myelodysplastic syndromes (MDS) in frequencies that vary between 9% and 40% depending on the techniques used in analysis. Gene amplification and direct sequencing provides the only unambiguous method of detecting those mutations that induce amino acid alterations. Using this technique, we analyzed 21 MDS patients for mutations in exon- 1 of N-RAS. Codon 12 mutations substituting aspartic acid (GAT) for glycine (GGT) were found in four cases, and a codon 13 mutation substituting alanine (GCT) for glycine (GGT) was detected in one patient. We conclude that N-RAS exon-1 mutations in one patient. We conclude that N-RAS exon-1 mutations producing amino acid changes occur in about 20% to 25% of MDS cases.     

Patterns of nucleotide misincorporations during enzymatic amplification and direct large-scale sequencing of ancient DNA

Whereas evolutionary inferences derived from present-day DNA sequences are by necessity indirect, ancient DNA sequences provide a direct view of past genetic variants. However, base lesions that accumulate in DNA over time may cause nucleotide misincorporations when ancient DNA sequences are replicated. By repeated amplifications of mitochondrial DNA sequences from a large number of ancient wolf remains, we show that C/G-to-T/A transitions are the predominant type of such misincorporations. Using a massively parallel sequencing method that allows large numbers of single DNA strands to be sequenced, we show that modifications of C, as well as to a lesser extent of G, residues cause such misincorporations. Experiments where oligonucleotides containing modified bases are used as templates in amplification reactions suggest that both of these types of misincorporations can be caused by deamination of the template bases. New DNA sequencing methods in conjunction with knowledge of misincorporation processes have now, in principle, opened the way for the determination of complete genomes from organisms that became extinct during and after the last glaciation.     

Whole-genome haplotyping by dilution,amplification, and sequencing

Standard whole-genome genotyping technologies are unable to determine haplotypes. Here we describe a method for rapid and cost-effective long-range haplotyping. Genomic DNA is diluted and distributed into multiple aliquots such that each aliquot receives a fraction of a haploid copy. The DNA template in each aliquot is amplified by multiple displacement amplification, converted into barcoded sequencing libraries using Nextera technology, and sequenced in multiplexed pools. To assess the performance of our method, we combined two male genomic DNA samples at equal ratios, resulting in a sample with diploid X chromosomes with known haplotypes. Pools of the multiplexed sequencing libraries were subjected to targeted pull-down of a 1-Mb contiguous region of the X-chromosome Duchenne muscular dystrophy gene. We were able to phase the Duchenne muscular dystrophy region into two contiguous haplotype blocks with a mean length of 494 kb. The haplotypes showed 99% agreement with the consensus base calls made by sequencing the individual DNAs. We subsequently used the strategy to haplotype two human genomes. Standard genomic sequencing to identify all heterozygous SNPs in the sample was combined with dilution-amplification–based sequencing data to resolve the phase of identified heterozygous SNPs. Using this procedure, we were able to phase >95% of the heterozygous SNPs from the diploid sequence data. The N50 for a Yoruba male DNA was 702 kb whereas the N50 for a European female DNA was 358 kb. Therefore, the strategy described here is suitable for haplotyping of a set of targeted regions as well as of the entire genome.     

Detection of ras gene mutations of primary hepatic malignant tumors by polymerase chain reaction and direct sequencing method

Ras genes are converted to active oncogenes by point mutations occurring in either codon 12, 13 or 61. We used polymerase chain reaction and direct sequence method for the analysis of these mutations. We examined 13 hepatocellular carcinomas, 8 cholangiocarcinomas, 2 hepatoblastomas and 1 biliary cystadenocarcinoma. Of these tumors, ras gene mutations were detected solely in cholangiocarcinomas. Cholangiocarcinoma showed a high incidence of K-ras gene mutation. Among 8 patients with cholangiocarcinoma, the mutation was detected at codon 12 in 4 and at codon 61 in 1. The incidence of K-ras gene mutation was especially high in the hilar type of cholangiocarcinoma as compared with the peripheral type.     

Detection of ultra-rare mutations by next-generation sequencing

Next-generation DNA sequencing promises to revolutionize clinical medicine and basic research. However, while this technology has the capacity to generate hundreds of billions of nucleotides of DNA sequence in a single experiment, the error rate of ～1% results in hundreds of millions of sequencing mistakes. These scattered errors can be tolerated in some applications but become extremely problematic when "deep sequencing" genetically heterogeneous mixtures, such as tumors or mixed microbial populations. To overcome limitations in sequencing accuracy, we have developed a method termed Duplex Sequencing. This approach greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors result in mutations in only one strand and can thus be discounted as technical error. We determine that Duplex Sequencing has a theoretical background error rate of less than one artifactual mutation per billion nucleotides sequenced. In addition, we establish that detection of mutations present in only one of the two strands of duplex DNA can be used to identify sites of DNA damage. We apply the method to directly assess the frequency and pattern of random mutations in mitochondrial DNA from human cells.     

The detection and use of hemoglobin mutants in the direct analysis of human globin genes

Many human globin-chain mutants contain amino acid replacements that result from single base changes in the corresponding globin gene. Using recombinants, the coding sequences of each of the alpha-, beta-, Ggamma- , and Agamma-globin genes have now been determined. Those sequences of DNA that are cleaved by a number of specific restriction endonucleases have been identified and accurately positioned. Mutations at these sequences abolish the restriction site, and therefore, the pattern of DNA fragments containing hybridizing globin-gene sequences is altered compared to DNA from normal persons. This allows the identification of one of a pair of cross-hybridizing human globin-gene sequences, as is shown here for the two alpha-globin, the two gamma-globin, and the delta- and beta-globin genes.     

Identification of mutations leading to the Lesch-Nyhan syndrome by automated direct DNA sequencing of in vitro amplified cDNA.

The Lesch-Nyhan (LN) syndrome is a severe X chromosome-linked disease that results from a deficiency of the purine salvage enzyme hypoxanthine phosphoribosyltransferase (HPRT). The mutations leading to the disease are heterogeneous and frequently arise as de novo events. We have identified nucleotide alterations in 15 independently arising HPRT-deficiency cases by direct DNA sequencing of in vitro amplified HPRT cDNA. We also demonstrate that the direct DNA sequence analysis can be automated, further simplifying the detection of new mutations at this locus. The mutations include DNA base substitutions, small DNA deletions, a single DNA base insertion, and errors in RNA splicing. The application of these procedures allows DNA diagnosis and carrier identification by the direct detection of the mutant alleles within individual families affected by LN.     

Melioidosis of the spleen diagnosed by 16S rDNA amplification and sequencing

We present the case of a 51-y-old male with multiple splenic abscesses that failed to resolve despite antimicrobial therapy targeted at Burkholderia pseudomallei. Elective splenectomy was performed to clarify the diagnosis. Pus from the spleen was subjected to universal bacterial 16S rDNA amplification and sequencing. The sequence obtained was 99% identical to 16S rDNA sequences for Burkholderia pseudomallei. Sequencing of the 16S rDNA is a very promising diagnostic tool allowing quick identification of Burkholderia pseudomallei directly from clinical specimen.     

Coupled amplification and sequencing of genomic DNA.

Addition of dideoxyribonucleotides during the exponential phase of the PCR should result in the synthesis of two complementary sequence ladders. We have explored this hypothesis to develop coupled amplification and sequencing of genomic DNA. Coupled amplification and sequencing is a biphasic method for sequencing both strands of template as they are amplified. Stage I selects and amplifies a single target from the genomic DNA sample. Stage II accomplishes the sequencing as well as additional amplification of the target using aliquots from the stage I reaction mixed with end-labeled primer and dideoxynucleotides. We have successfully applied coupled amplification and sequencing to a 300-base-pair fragment 4 kilobases upstream from HOX2B directly from human whole genomic DNA.     

Patterns of K-ras codon 12 and 13 mutations found in pancreatic adenocarcinoma of 30 Chinese patients by microdissection, PCR and direct sequencing

BACKGROUND AND AIM: To our knowledge there are few reports on the K-ras mutation pattern of pancreatic adenocarcinoma from Chinese mainland patients. We examined surgically resected formalin-fixed, paraffin-embedded primary pancreatic adenocarcinoma tissue blocks for the presence of activating point mutations at codon 12 and 13 of the K-ras gene. METHODS: Mutations were detected through the use of microdissection, polymerase chain reaction (PCR) and direct sequencing. The results were confirmed by reverse sequencing. RESULTS: The combination of microdissection, PCR and direct sequencing techniques resulted in a rapid and sensitive detection of K-ras mutations at codon 12 and 13. Twenty-five (83%) of the 30 pancreatic adenocarcinomas examined harbored K-ras mutation. Among the 25 pancreatic adenocarcinomas, 24 showed K-ras mutation at codon 12 (11 with GGT-GTT, seven with GGT-GAT, four with GGT-CGT, and two with GGT-TGT), and only one showed a GGC-TGC mutation at codon 13. In this study most of K-ras mutations at codon 12 were at the second base (72%, 18/25) with a transition/transversion ratio of 1 : 1.57 (7/11). CONCLUSIONS: The mutation profiles of K-ras at codon 12 in our pancreatic adenocarcinoma samples were significantly different from those of European and Japanese samples.     

Patterns of K-ras codon 12 and 13 mutations found in pancreatic adenocarcinoma of 30 Chinese patients by microdissection, PCR and direct sequencing

Background and Aim:  To our knowledge there are few reports on the K-ras mutation pattern of pancreatic adenocarcinoma from Chinese mainland patients. We examined surgically resected formalin-fixed, paraffin-embedded primary pancreatic adenocarcinoma tissue blocks for the presence of activating point mutations at codon 12 and 13 of the K-ras gene.
Methods:  Mutations were detected through the use of microdissection, polymerase chain reaction (PCR) and direct sequencing. The results were confirmed by reverse sequencing.
Results:  The combination of microdissection, PCR and direct sequencing techniques resulted in a rapid and sensitive detection of K-ras mutations at codon 12 and 13. Twenty-five (83%) of the 30 pancreatic adenocarcinomas examined harbored K-ras mutation. Among the 25 pancreatic adenocarcinomas, 24 showed K-ras mutation at codon 12 (11 with GGT-GTT, seven with GGT-GAT, four with GGT-CGT, and two with GGT-TGT), and only one showed a GGC-TGC mutation at codon 13. In this study most of K-ras mutations at codon 12 were at the second base (72%, 18/25) with a transition/transversion ratio of 1 : 1.57 (7/11).
Conclusions:  The mutation profiles of K-ras at codon 12 in our pancreatic adenocarcinoma samples were significantly different from those of European and Japanese samples.     

Known and new delta globin gene mutations and their diagnostic significance

Mutations in the delta-globin gene (HBD, MIM# 142000) are not pathologically relevant. However, since high HbA2 levels are diagnostic for beta-thalassemia trait and a lowered level for an alpha- or delta-mutation, co-inheritance of delta- and beta-gene defects may lead to misinterpretation of diagnostic results. We examined 29 cases with low HbA2 level diagnosed in our laboratory, in the presence or absence of a second HbA2 fraction. We found a delta globin gene mutation in 20 cases. In total four different known mutations were found, three structural and one expressional. Moreover, two new defects were observed, one causing a structural abnormality and one a beta-thalassemia. The structural abnormality HBD c.431A->G (p.His144Arg)(dcd 143 CAC->CGC) was homologous to the beta-globin gene variant called Hb-Abruzzo and we have named this mutation HbA2 -Abruzzo. The new delta-thalassemia defect HBD c.-118C->T (d -68 C->T) has no homology on the beta-globin gene (HBB, MIM# 141900). All mutations caused a low HbA2 level and through this could lead to misdiagnosis when inherited together with a beta-thalassemia.     

Identification of the cause of a brain abscess by direct 16S ribosomal DNA sequencing

We report the case of a young man who apparently suffered successive episodes of meningitis and cerebral abscess over a 1-month period, both of which were diagnosed by two different molecular approaches; PCR for Neisseria meningitidis IS1106 from CSF and 16S rRNA gene sequencing on a specimen of brain pus. In each case, cultures were negative due to prior antibiotic therapy.     

An immunoassay to detect human embryonic epsilon globin chains by a murine monoclonal antibody

Human embryonic epsilon globin chains were isolated from lysates of cultured human leukemic K562 cells induced with hemin. Mice were immunized with epsilon globin chains, and a murine hybridoma cell line was established that secreted monoclonal anti-epsilon globin antibody. A sensitive and specific slot blot immunoassay for epsilon globin chains has been developed. epsilon Globin chains were shown to be present in hemolysates from fetuses of 20 weeks' gestation but were not detected in normal cord blood hemolysates. This immunoassay is useful for the study of epsilon globin ontogeny during embryonic and fetal development as well as for searching for inappropriate epsilon globin chain expression in hematologic disorders resulting from genetic abnormalities or neoplastic processes.