Short Stretches Of DNA Research Articles

Predicting the in vivo signature of human gene regulatory sequences

In the living cell nucleus, genomic DNA is packaged into chromatin. DNA sequences that regulate transcription and other chromosomal processes are associated with local disruptions, or 'openings', in chromatin structure caused by the cooperative action of regulatory proteins. Such perturbations are extremely specific for cis-regulatory elements and occur over short stretches of DNA (typically approximately 250 bp). They can be detected experimentally as DNaseI hypersensitive sites (HSs) in vivo, though the process is extremely laborious and costly. The ability to discriminate DNaseI HSs computationally would have a major impact on the annotation and utilization of the human genome. We found that a supervised pattern recognition algorithm, trained using a set of 280 DNaseI HS and 737 non-HS control sequences from erythroid cells, was capable of de novo prediction of HSs across the human genome with surprisingly high accuracy determined by prospective in vivo validation. Systematic application of this computational approach will greatly facilitate the discovery and analysis of functional non-coding elements in the human and other complex genomes. Supplementary data is available at noble.gs.washington.edu/proj/hs

DNA twisting flexibility and the formation of sharply looped protein–DNA complexes

Gene-regulatory complexes often require that pairs of DNA-bound proteins interact by looping-out short (often approximately 100-bp) stretches of DNA. The loops can vary in detailed length and sequence and, thus, in total helical twist, which radically alters their geometry. How this variability is accommodated structurally is not known. Here we show that the inherent twistability of 89- to 105-bp DNA circles exceeds theoretical expectation by up to 400-fold. These results can be explained only by greatly enhanced DNA flexibility, not by permanent bends. They invalidate the use of classic theories of flexibility for understanding sharp DNA looping but support predictions of two recent theories. Our findings imply an active role for DNA flexibility in loop formation and suggest that variability in the detailed helical twist of regulatory loops is accommodated naturally by the inherent twistability of the DNA.

Polymerase Chain Reaction in the Diagnosis of Uveitis

Polymerase chain reaction (PCR) is a technique involving enzymatic amplification of nucleic acid sequences in repeated cycles of denaturation, oligonucleotide annealing, and DNA polymerase extension.1,2 The PCR uses in vitro enzymatic synthesis to amplify specific DNA sequences within a few hours. Since its inception in 1985, PCR has revolutionized research in the biologic sciences and medicine and has influenced criminology and law.3 The inventor of PCR, Kary Mullis, was awarded the Nobel Prize in Chemistry in 1993 in recognition of the extraordinary impact of PCR technology on scientific research.4,5 Polymerase chain reaction consists of repetitive cycles of specific DNA synthesis, defined by short stretches of preselected DNA. With each cycle, there is a doubling of the final, desired DNA product such that a million-fold amplification is possible.6 This powerful technique has numerous applications in diagnostic pathology, especially in the fields of microbiology, genetics, and oncology. Polymerase chain reaction has been used to diagnose uveitis, including viral uveitis, mycobacterial intraocular infections, infectious endophthalmitis, and protozoa eye diseases.7 However, the extremely high sensitivity of PCR can produce false-positive results, whereas its high specificity may produce false-negative results. These pitfalls can be minimized by techniques such as the use of both positive and negative controls, real-time PCR, and the performance of tests in an experienced laboratory. In any case, to ensure an accurate diagnosis, one must consider clinical data in the interpretation of a PCR result. We present diagnostic applications and examples of utilization of PCR in infectious and noninfectious uveitis, as well as in masquerade syndromes and other common ocular diseases with inflammatory components.

Aberrant Somatic Hypermutation in Primary Mediastinal Large B-Cell Lymphoma.

Abstract Primary mediastinal large B-cell lymphoma (PMLBCL) is recognised as a subtype of diffuse large B-cell lymphoma (DLBCL) arising in the mediastinum. With respect to DLBCL, PMLBCL displays specific clinical, molecular and morphological features suggesting that PMLBCL may represent a distinct clinico-pathologic entity. Aberrant somatic hypermutation (SHM) of PIM-1, PAX-5, RhoH/TTF and c-MYC has been advocated as a molecular feature distinctive of DLBCL. To investigate wether the same mechanism is associated with PMLBCL, we performed mutational analysis of PIM-1, PAX-5, RhoH/TTF and c-MYC in a panel of 19 PMLBCL. For comparison, 19 DLBCL were also analysed. For each gene, a region previously shown to contain >90% of mutations was analysed by PCR amplification and DNA direct sequencing. Overall, the prevalence of mutated cases was similar among DLBCL and PMLBCL. Mutations targeting at least one of the 4 genes were found in 14/19 (73.6%) PMLBCL and 13/19 (68.4%) DLBCL, while mutations in more than one gene were found in 7/19 (36.8%) PMLBCL and 9/19 (47.3%) DLBCL. Among the four genes, the prevalence of mutation and the mutation frequency was superimposable between PMLBCL and DLBCL. In fact, PAX-5 was mutated in 9/19 (47.3%) PMLBCL with a mean mutation frequency of 0.20 x 10−2/bp and in 7/19 (36.8%) DLBCL with a mean mutation frequency of 0.18 x 10−2/bp; RhoH/TTF was mutated in 6/19 (31.5%) PMLBCL with a mean mutation frequency of 0.08 x 10−2/bp and in 8/19 (42.1%) DLBCL with a mean mutation frequency of 0.27 x 10−2/bp; PIM-1 was mutated in 3/19 (15.7%) PMLBCL with a mean mutation frequency of 0.09 x 10−2/bp and in 7/19 (36.8%) DLBCL with a mean mutation frequency of 0.11 x 10−2/bp; c-MYC was mutated in 6/19 (31.5%) PMLBCL with a mean mutation frequency of 0.23 x 10−2/bp and in 5/19 (26.3%) DLBCL with a mean mutation frequency of 0.11 x 10−2. The mutation pattern was also similar between PMLBCL and DLBCL and was consistent with the SHM process. A total of 74 mutational events were detected in PMLBCL. The majority of mutations were represented by single base-pair substitution (n=66), whereas only 8 deletions of a short DNA stretch were observed. Of the 66 single base-pair substitutions, 41 were transitions and 25 were transversions, with a transition/transversion ratio of 1.64 and a G+C/A+T ratio of 3.6. Eleven out of 66 (16.6%) single base-pair substitutions felt within RGYW/WRCY motifs. Among DLBCL a total of 87 mutational events were detected. Mutations were preferentially represented by single base-pair substitutions (n=81), whereas only 4 deletions and 2 insertions of a short DNA stretch were observed. Of the 81 single base-pair substitutions, 42 were transitions and 39 were transversions, with a transition/transversion ratio of 1.07 and a G+C/A+T ratio of 1.89. Twenty six out of 81 (32.1%) single base-pair substitutions felt within RGYW/WRCY motifs. The implication of our results are twofold. First, aberrant SHM is involved in the pathogenesis of PMLBCL. Second, our results indicate that aberrant SHM targets both PMLBCL and DLBCL with similar prevalence, distribution and mutation pattern. Since aberrant SHM has been advocated as a molecular marker of DLBCL, our results corroborate the notion that PMLBCL represent a subtype of DLBCL rather than a distinct clinico-pathologic entity.

Histogenesis and Pathogenesis of Primary Breast Lymphoma.

Abstract Primary lymphoma of the breast accounts for <2% of extranodal non-Hodgkin’s lymphoma and generally displays morphology consistent with diffuse large B-cell lymphoma (DLBCL). The pathogenesis and histogenesis of primary breast lymphoma are currently unknown and represented the aim of this study. Thirteen cases of primary breast lymphoma (all DLBCL) were analyzed for physiological somatic hypermutation (SHM) of IgV and BCL-6 genes, as well as for aberrant SHM of PAX-5, PIM-1, RhoH/TTF and c-MYC genes. A functional IgVH rearrangement was identified in 8/13 (61.5%) primary breast lymphomas. All IgVH genes displayed SHM, with a mutation frequency ranging from 4.0% to 25.9%, suggesing that primary breast lymphomas derive from GC-related B-cells. The IgVH gene families utilized included VH4 (4/8 cases), VH3 (3/8 cases) and VH2 (1/8 case). Three cases utilized the same VH 4.30.1/4-31 gene. Mutations of BCL-6 were detected in 7/13 (53.8%) cases, further confirming the finding that primary lymphomas of the breast derive from GC-experienced B cells. Analysis of aberrant SHM of proto-oncogenes was performed on selected regions known to contain >90% of mutations found in lymphoma. Overall, mutations in at least one of the four proto-oncogenes targeted by aberrant SHM were found in 9/13 (69%) primary breast lymphomas, whereas mutations in more than one gene were found in 4/9 (44%) cases. Each of the four proto-oncogenes was altered in a significant fraction of primary breast lymphomas (PAX-5 in 4/9 cases; RhoH/TTF in 5/9 cases; PIM-1 in 5/9 cases and c-MYC in 2/9 cases). The overwhelming majority of mutations was represented by single base-pair substitutions (n=36), whereas in only one instance a deletion of a short DNA stretch was observed. Among the 36 single base-pair substitutions detected in primary breast lymphoma, the transition/transversion ratio was 1.76 (expected 0.5; p<0.01; Chi square test), reflecting the mutational profile seen in nodal DLBCL of immunocompetent hosts. In PIM-1, a fraction of mutations led to amino acid substitution, with potential functional consequences. In particular, three primary breast lymphomas displayed four missense mutations localized within the serine-threonine kinase domain of PIM-1. The association of primary breast lymphoma with aberrant SHM of proto-oncogenes expands the types of aggressive lymphomas marked by this molecular abnormality and provides clues for understanding breast lymphoma pathogenesis. In particular, missense mutations in the PIM-1 coding region can deregulate its function, whereas mutations of the 5′ regulatory regions of PAX-5, RhoH/TTF and c-MYC are expected to influence the expression and regulation of these genes in a fashion similar to that reported for the BCL-6 gene in B-cell lymphoma. Consistent with the role of PAX-5 in B-cell differentiation, of RhoH/TTF in signal transduction, and of c-MYC in B-cell growth and fate, deregulation of these genes by aberrant hypermutation may contribute to breast lymphoma pathogenesis by multiple pathways.

Obstacle Bypass in Protein Motion along DNA by Two-dimensional Rather than One-dimensional Sliding

Site-specific DNA-binding proteins locate their target sites by facilitated diffusion. Several proteins have been shown to slide along DNA in vitro. However, whereas sliding is often envisaged as one-dimensional tracking of the DNA major groove, such a mechanism would not allow linear diffusion over long distances in vivo, where short stretches of free DNA are delimited by bound proteins. I propose a two-dimensional sliding mechanism, in which the protein diffuses freely on the cylindrical DNA surface, and I present experiments that can distinguish between one- and higher-dimensional diffusion along the DNA contour length. At 100 mm NaCl, translocation of EcoRI restriction endonuclease between sites on two DNA helices connected by a Holliday junction is as efficient as between sites on the same helix, indicating a three-dimensional mechanism. At 25 mm NaCl, translocation between sites on the same DNA helix is more efficient, indicating a role for sliding at low ionic strength. Obstacles attached to the major groove of one face of the DNA helix did not interfere with sliding, regardless of their orientation relative to the cleavage sites. This result is compatible with two-dimensional but not one-dimensional sliding. As illustrated by Monte-Carlo simulation, two-dimensional sliding may not only allow proteins to move around nucleosomes in vivo but also reduce the redundancy of their search for the target site.

Split-Marker Recombination for Efficient Targeted Deletion of Fungal Genes

A commonly used method for fungal gene deletion is introduction of linear DNA consisting of a selectable marker gene flanked on both sides by short stretches of DNA that target a gene of interest (W irsel et al 1996 Curr. Genet 29:241-249). Gene deletion in Cochliobolus heterostrophus and Gibberella zeae occurs efficiently with this approach. To facilitate deletion construct synthesis, we have applied the split-marker” deletion strategy previously developed for Saccharomyces cerevisiae (Fairhead et al. 1996 Y east 12:1439-57; Fairhead et al. 1998 Gene 223:33-46). Here, we describe both fusion PCR-based and plasmid-based deletion methods using this strategy with PEG-mediated protoplast transformation (Turgeon et al, 1985 M ol. Gen. Genet. 201:450-453). These methods are predicted to work well with any transformable fungus that undergoes homologous recombination between chromosomal and introduced DNA sequences.

Multiple group-specific sequencing primers for reliable and rapid DNA sequencing

Pyrosequencing technology is a bioluminometric DNA sequencing method that employs a cascade of four enzymes to deliver sequence signals. To date this technology has been limited to the sequencing of short stretches of DNA. As an improvement to this technique, we have introduced a bacterial group-specific, multiple sequencing primer approach that circumvents sequencing of less informative semi-conservative regions of the 16S rRNA gene. This new approach is suitable for challenging templates, improving sequence data quality, avoiding sequencing of non-specific amplification products, lessening sequencing time, and moreover, this strategy should open the way for many new applications in the future. The group-specific, multiple sequencing primers can be applied in the Sanger dideoxy sequencing method as well. In addition, we have improved the chemistry of the Pyrosequencing system enabling sequencing of longer stretches of DNA, which allows numerous new applications.

Drug discovery with engineered zinc-finger proteins.

Zinc-finger proteins (ZFPs) that recognize novel DNA sequences are the basis of a powerful technology platform with many uses in drug discovery and therapeutics. These proteins have been used as the DNA-binding domains of novel transcription factors (ZFP TFs), which are useful for validating genes as drug targets and for engineering cell lines for small-molecule screening and protein production. Recently, they have also been used as a basis for novel human therapeutics. Most of our advances in the design and application of these ZFP TFs rely on our ability to engineer ZFPs that bind short stretches of DNA (typically 9-18 base pairs) located within the promoters of target genes. Here, we summarize the methods used to design these DNA-binding domains, explain how they are incorporated into novel transcription factors (and other useful molecules) and describe some key applications in drug discovery.

The Structure of DNA within Cationic Lipid/DNA Complexes

The structure of DNA within CLDCs used for gene delivery is controversial. Previous studies using CD have been interpreted to indicate that the DNA is converted from normal B to C form in complexes. This investigation reexamines this interpretation using CD of model complexes, FTIR as well as Raman spectroscopy and molecular dynamics simulations to address this issue. CD spectra of supercoiled plasmid DNA undergo a significant loss of rotational strength in the signal near 275 nm upon interaction with either the cationic lipid dimethyldioctadecylammonium bromide or 1,2-dioleoyltrimethylammonium propane. This loss of rotational strength is shown, however, by both FTIR and Raman spectroscopy to occur within the parameters of the B-type conformation. Contributions of absorption flattening and differential scattering to the CD spectra of complexes are unable to account for the observed spectra. Model studies of the CD of complexes prepared from synthetic oligonucleotides of varying length suggest that significant reductions in rotational strength can occur within short stretches of DNA. Furthermore, some alteration in the hydrogen bonding of bases within CLDCs is indicated in the FTIR and Raman spectroscopy results. In addition, alterations in base stacking interactions as well as hydrogen bonding are suggested by molecular dynamics simulations. A global interpretation of all of the data suggests the DNA component of CLDCs remains in a variant B form in which base/base interactions are perturbed.

PCR-SSCP analysis of polymorphism: a simple and sensitive method for detecting differences between short segments of DNA.

AbstractPolymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) (1) is a simple method that allows one to rapidly determine whether there are sequence differences between relatively short stretches of DNA. Coupled with sequence analysis, SSCP is an extremely useful method for both identifying and characterizing genetic polymorphisms and mutations. The theory of SSCP is that the primary sequence and the length of a single stranded DNA fragment determine its conformation when it is resolved in a nondenaturing polyacrylamide gel. Even single-base differences can cause different secondary conformations and thus result in different migration rates of the DNA strands. Radioactive nucleotides are incorporated into the DNA strands by PCR, making it possible to detect the DNA by autoradiography. SSCP has been widely used to identify mutations in host genes such as p53 (2–5) and in viruses, such as simian immuno-deficiency virus (SIV), during the course of infection (6). SSCP has been used to identify and characterize polymorphisms in a variety of genes (7–9) and was effective in characterizing alleles of linked genes present in individual sperm (10).KeywordsAmmonium PersulphateCD1a GeneSSCP PatternIndividual SpermMigration DifferenceThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Ectoderm gene activation in sea urchin embryos mediated by the CCAAT-binding factor

Transcriptional enhancers are short stretches of DNA that function to achieve highly specific patterns of gene expression. To identify the mechanisms by which enhancers achieve their specificity, we made use of an enhancer from the aboral ectoderm-specific spec2a gene of the sea urchin Strongylocentrotus purpuratus. The spec2a enhancer contains five cis-regulatory elements within 78 base pairs that interact with five distinct DNA-binding proteins to confer aboral ectoderm expression. Here, we present an analysis of the sea urchin CCAAT binding factor (CBF), which binds to a CCAAT motif within the spec2a enhancer. S. purpuratus CBF and SpOtx, a ubiquitously expressed factor, act together at closely placed cis-regulatory elements to mediate spec2a transcription in the ectoderm. SpCBF was the sole factor that bound to the spec2a CCAAT element, and two of the three subunits that make up the CBF holoprotein were cloned and shown to have high sequence conservation with their vertebrate orthologs. Based on its involvement in the regulation of several other sea urchin genes, SpCBF appears to be a major transcription factor in the sea urchin embryo for positive regulation of ectoderm gene expression. In addition to its role in vertebrate cell growth and proliferation, our results indicate that CBF also functions at the early stages of germ layer formation, namely ectoderm differentiation.

Long-Read Pyrosequencing Using Pure 2′-Deoxyadenosine-5′-O′-(1-thiotriphosphate) Sp-Isomer

Pyrosequencing, a nonelectrophoretic DNA sequencing method that uses a luciferase-based enzymatic system to monitor DNA synthesis in real time, has so far been limited to sequencing of short stretches of DNA. To increase the signal-to-noise ratio in pyrosequencing the natural dATP was replaced by dATPalphaS (M. Ronaghi et al., 1996, Anal. Biochem. 242, 84-89). The applied dATPalphaS was a mixture of two isomers (Sp and Rp). We show here that by the introduction of pure 2'-deoxyadenosine-5'-O'-(1-thiotriphosphate) Sp-isomer in pyrosequencing substantial longer reads could be obtained. The pure Sp-isomer allowed lower nucleotide concentration to be used and improved the possibility to read through poly(T) regions. In general, a doubling of the read length could be obtained by the use of pure Sp-isomer. Pyrosequencing data for 50 to 100 bases could be generated on different types of template. The longer read will enable numerous new applications, such as identification and typing of medically important microorganisms as well as resequencing of DNA fragments for mutation screening and clone checking.

Methylation-sensitive restriction fingerprinting.

Methylation of cytosine residues is an almost ubiquitous finding of higher organisms (). The majority of this methylation occurs at the dinucleotide CpG (where p denotes a phosphate group) (). CpG sites are distributed throughout the genome with clusters of the sequence being found in the 5′ promoter region of housekeeping genes, in groups known as CpG islands. These short stretches of DNA have a have a C and G base composition, which in mammals and avians is estimated to be 10 times higher than in bulk DNA ()

Free-energy component analysis of 40 protein-DNA complexes: a consensus view on the thermodynamics of binding at the molecular level.

Noncovalent association of proteins to specific target sites on DNA--a process central to gene expression and regulation--has thus far proven to be idiosyncratic and elusive to generalizations on the nature of the driving forces. The spate of structural information on protein--DNA complexes sets the stage for theoretical investigations on the molecular thermodynamics of binding aimed at identifying forces responsible for specific macromolecular recognition. Computation of absolute binding free energies for systems of this complexity transiting from structural information is a stupendous task. Adopting some recent progresses in treating atomic level interactions in proteins and nucleic acids including solvent and salt effects, we have put together an energy component methodology cast in a phenomenological mode and amenable to systematic improvements and developed a computational first atlas of the free energy contributors to binding in approximately 40 protein-DNA complexes representing a variety of structural motifs and functions. Illustrating vividly the compensatory nature of the free energy components contributing to the energetics of recognition for attaining optimal binding, our results highlight unambiguously the roles played by packing, electrostatics including hydrogen bonds, ion and water release (cavitation) in protein-DNA binding. Cavitation and van der Waals contributions without exception favor complexation. The electrostatics is marginally unfavorable in a consensus view. Basic residues on the protein contribute favorably to binding despite the desolvation expense. The electrostatics arising from the acidic and neutral residues proves unfavorable to binding. An enveloping mode of binding to short stretches of DNA makes for a strong unfavorable net electrostatics but a highly favorable van der Waals and cavitation contribution. Thus, noncovalent protein-DNA association is a system-specific fine balancing act of these diverse competing forces. With the advances in computational methods as applied to macromolecular recognition, the challenge now seems to be to correlate the differential (initial vs. final) energetics to substituent effects in drug design and to move from affinity to specificity.

P16 Ink4a and p19 Arf: terrible twins

p16Ink4a and p19Arf might be considered to be molecular twins. They are born from the same genetic locus CDKN2A (INK4a/ARF) and have tricked biologists for some years about which one is important in preventing cancer. Now cancer biologists have nailed down this terrible twosome and formally proved that both are crucial for inhibiting tumorigenesis. The best way to ‘prove’ that a gene is a tumour suppressor is to mutate the gene in the mouse and show subsequent tumour susceptibility. So, although it will not come as a surprise to learn that mice lacking p16Ink4a form tumours, it is a relief to cancer biologists to have p16Ink4a officially declared a tumour suppressor gene together with his infamous twin p19Arf (1xLoss of p16Ink4a confers susceptibility to metastatic melanoma in mice. Krimpenfort, P. et al. Nature. 2001; 413: 83–86Crossref | PubMed | Scopus (394)See all References, 2xLoss of p16Ink4a with retention of p19Arf predispose mice to tumorigenesis. Sharpless, N.E. et al. Nature. 2001; 413: 86–91Crossref | PubMed | Scopus (549)See all References).The two sibling proteins are encoded at one of the most remarkable loci in the human genome – one short stretch of DNA that generates two overlapping transcripts that encode two structurally unrelated proteins, both of which inhibit the cell cycle. p16Ink4a inhibits cyclin-dependent kinases CDK4 and CDK6, thereby regulating the retinoblastoma pRB pathway. By contrast, p19Arf (called p14Arf in humans) achieves cell-cycle arrest by stabilizing the p53 protein. This is biology at its most elegant. Given their ability to regulate both pRb and p53 checkpoint pathways, it's hardly surprising that the CDK2A-INK4A/ARF locus is mutated, or silenced epigenetically, in many human tumours. Although some of these mutations knock out both proteins, others affect just one of the pair, implying that both might behave as independent tumour suppressors. Researchers have turned to genetically engineered mice to solve the riddle of the tumour suppressor twins.Several years ago Kamijo et al.3xTumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF. Kamijo, T. et al. Cell. 1997; 91: 649–659Abstract | Full Text | Full Text PDF | PubMedSee all References3 showed that mice lacking p19Arf develop tumours early in life, demonstrating that p19Arf is a bona fide tumour suppressor. Now two independent groups (Krimpenfort et al.1xLoss of p16Ink4a confers susceptibility to metastatic melanoma in mice. Krimpenfort, P. et al. Nature. 2001; 413: 83–86Crossref | PubMed | Scopus (394)See all References1 and Sharpless et al.2xLoss of p16Ink4a with retention of p19Arf predispose mice to tumorigenesis. Sharpless, N.E. et al. Nature. 2001; 413: 86–91Crossref | PubMed | Scopus (549)See all References2) have engineered mice with mutations so that they express normal p19Arf but no p16Ink4a. These mutant animals developed normally and the adult mice are quite perky, except for some slight increases in thymocyte numbers. Surprisingly, fibroblasts derived from these animals behaved quite normally too, in terms of proliferation, senescence or Ras-transformation. This is in marked contrast to the abnormal growth of fibroblasts lacking p19Arf.But as the p16Ink4a-null mice grew older, some of them developed tumours including soft-tissue sarcomas, lymphomas and melanomas. When the researchers looked at carcinogen-induced malignancy, both groups found that the mutation dramatically affected susceptibility. Mice treated with the carcinogen 7,12-dimethylbenzanthracene (DMBA) developed lung adenomas, soft-tissue carcinomas, skin papillomas and melanomas. Melanomas are often found in human patients with germline mutations in the CDKN2A locus. Furthermore, the researchers found that combining mutations in p19Arf and p16Ink4a leads to an increase in the number and range of spontaneous tumours. These studies offer an animal model to explore the role of the CDKN2A locus in human malignancies. Twins tend to get blamed as a pair. It looks like each of these talented twins plays a distinct role in tumour suppression.

A Mediaeval Case of Lepromatous Leprosy from 13–14th Century Orkney, Scotland

Erosion in the 1960s resulted in exposure of human skeletal remains from a Norse Christian cemetery at Newark Bay, Orkney, Scotland. One set of remains showed osteological evidence of advanced lepromatous leprosy, but the absence of bones from the lower limbs precluded definitive diagnosis. The aim of the present study was to determine whether Mycobacterium leprae could be detected in bone extracts, as a means of confirming the diagnosis of leprosy. Bone samples were examined from the suspected leprosy case and from a second contemporary burial thought to be free of disease. DNA was amplified by polymerase chain reaction (PCR) using primers specific for a repetitive element (RLEP) characteristic of M. leprae. Additional PCR tests specific for Mycobacterium tuberculosis and for amelogenin (a human gene suitable for sex determination) were also applied to the samples. M. leprae DNA was detected only in the skull sample from the suspected leprosy case. The DNA sequence was identical to that found in present day isolates of M. leprae. Positive results were obtained only using a PCR reaction designed to amplify relatively short stretches of DNA (<175 bp), suggesting the microbial DNA had undergone extensive fragmentation. There was no evidence of M. tuberculosis DNA in bones from the leprosy suspect or control individual. The ability to recover ancient samples of DNA provides an opportunity to study long-term evolutionary changes that may affect the epidemiology of microbial pathogens.

Antisense Inhibition of the Renin–Angiotensin System in Brain and Peripheral Organs

Antisense inhibition is a method of attenuating the target at the gene expression level. There are two main groups of molecular tools for this goal. The first includes the use of short synthetic stretches of DNA-antisense oligodeoxynucleotides. The second tool is the use of vectors (plasmids or viruses) containing the gene of interest subcloned in the antisense orientation, which in the cells produces the antisense RNA. Both antisense DNA and RNA can bind to the complementary sense mRNA and interfere with its translation. Effects are usually short lasting (days) for oligodeoxynucleotides and longer lasting (weeks or months) for vectors. In this article we briefly describe techniques of antisense inhibition in the context of the renin-angiotensin system.

Rapid determination of short DNA sequences by the use of MALDI-MS.

We have developed a protocol for rapid sequencing of short DNA stretches (15-20 nt) using MALDI-TOF-MS. The protocol is based on the Sanger concept with the modification that double-stranded template DNA is used and all four sequencing reactions are performed in one reaction vial. The sequencing products are separated and detected by MALDI-TOF-MS and the sequence is determined by comparing measured molecular mass differences to expected values. The protocol is optimized for low costs and broad applicability. One reaction typically includes 300 fmol template, 10 pmol primer and 200 pmol each nucleotide monomer. Neither the primer nor any of the nucleotide monomers are labeled. Solid phase purification, concentration and mass spectrometric sample preparation of the sequencing products are accomplished in a few minutes and parallel processing of 96 samples is possible. The mass spectrometric analyses and subsequent sequence read-out require only a few seconds per template.

A novel procedure for efficient genotyping of single nucleotide polymorphisms.

Due to the surge in interest in using single nucleotide polymorphisms (SNPs) for genotyping a facile and affordable method for this is an absolute necessity. Here we introduce a procedure that combines an easily automatable single tube sample preparation with an efficient high throughput mass spectrometric analysis technique. Known point mutations or single nucleotide polymorphisms are easily analysed by this procedure. It starts with PCR amplification of a short stretch of genomic DNA, for example an exon of a gene containing a SNP. By shrimp alkaline phosphatase digest residual dNTPs are destroyed. Allele-specific products are generated using a special primer, a conditioned set of alpha-S-dNTPs and alpha-S-ddNTPs and a fresh DNA polymerase in a primer extension reaction. Unmodified DNA is removed by 5'-phospho-diesterase digestion and the modified products are alkylated to increase the detection sensitivity in the mass spectrometric analysis. All steps of the preparation are simple additions of solutions and incubations. The procedure operates at the lowest practical sample volumes and in contrast to other genotyping protocols with mass spectrometric detection requires no purification. This reduces the cost and makes it easy to implement. Here it is demonstrated in a version using positive ion detection on described mutations in exon 17 of the amyloid precursor protein gene and in a version using negative ion detection on three SNPs of the granulocyte-macrophage colony stimulating factor gene. Preparation and analysis of SNPs is shown separately and simultaneously, thus demonstrating the multiplexibility of this genotyping procedure. The preparation protocol for genotyping is adapted to the conditions used for the SNP discovery method by denaturing HPLC, thus demonstrating a facile link between protocols for SNP discovery and SNP genotyping. Results corresponded unanimously with the control sequencing. The procedure is useful for high throughput genotyping as it is required for gene identification and pharmacogenomics where large numbers of DNA samples have to be analysed. We have named this procedure the 'GOOD Assay' for SNP analysis.