bluuebunny/crossref_metadata_2025 huggingface dataset:
Sensitive two-stage PCR of p53 genomic DNA exons 5-9.
Authors: Kusser W C , Levin D B , Glickman B W
Published: June 2007

To assess the status of the tumor suppressor gene p53 from samples with low levels (sub-nanogram amounts) of genomic DNA (e.g., from exfoliated cells, skin, small biopsies, mucosa), a technique based on two successive rounds of PCR was developed. In the first round, a 1.84-kb fragment spanning exons 5-9 was generated using a "touchdown" protocol. After purification by spun-column chromatography, this fragment was used as a template for the amplification of the individual exons 5-9 with inner primer sets specific for the adjacent intronic regions. Using this nested primer approach, several micrograms of each individual exon were obtained starting from as little as 62.5 pg of total genomic DNA. This material proved ideal for future analyses by single-strand conformational polymorphism (SSCP) analysis and DNA sequencing.
High-level expression, purification, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated form deficient in 5' to 3' exonuclease activity.
Authors: Lawyer F C , Stoffel S , Saiki R K , Chang S Y , Landre P A , Abramson R D , Gelfand D H
Published: June 2007

The Thermus aquaticus DNA polymerase I (Taq Pol I) gene was cloned into a plasmid expression vector that utilizes the strong bacteriophage lambda PL promoter. A truncated form of Taq Pol I was also constructed. The two constructs made it possible to compare the full-length 832-amino-acid Taq Pol I and a deletion derivative encoding a 544-amino-acid translation product, the Stoffel fragment. Upon heat induction, the 832-amino-acid construct produced 1-2% of total protein as Taq Pol I. The induced 544-amino-acid construct produced 3% of total protein as Stoffel fragment. Enzyme purification included cell lysis, heat treatment followed by Polymin P precipitation of nucleic acids, phenyl sepharose column chromatography, and heparin-Sepharose column chromatography. For full-length 94-kD Taq Pol I, yield was 3.26 x 10(7) units of activity from 165 grams wet weight cell paste. For the 61-kD Taq Pol I Stoffel fragment, the yield was 1.03 x 10(6) units of activity from 15.6 grams wet weight cell paste. The two enzymes have maximal activity at 75 degrees C to 80 degrees C, 2-4 mM MgCl2 and 10-55 mM KCl. The nature of the substrate determines the precise conditions for maximal enzyme activity. For both proteins, MgCl2 is the preferred cofactor compared to MnCl2, CoCl2, and NiCl2. The full-length Taq Pol I has an activity half-life of 9 min at 97.5 degrees C. The Stoffel fragment has a half-life of 21 min at 97.5 degrees C. Taq Pol I contains a polymerization-dependent 5' to 3' exonuclease activity whereas the Stoffel fragment, deleted for the 5' to 3' exonuclease domain, does not possess that activity. A comparison is made among thermostable DNA polymerases that have been characterized; specific activities of 292,000 units/mg for Taq Pol I and 369,000 units/mg for the Stoffel fragment are the highest reported.
Rapid characterization of HIV-1 sequence diversity using denaturing gradient gel electrophoresis and direct automated DNA sequencing of PCR products.
Authors: Andersson B , Ying J H , Lewis D E , Gibbs R A
Published: June 2007

A direct method for visualization and isolation of sequence variants of human immunodeficiency virus type 1 (HIV-1) utilizing denaturing gradient gel electrophoresis (DGGE) combined with automated direct DNA sequencing was developed. Two fragments from the env gene and one from the nef gene of HIV-1, which together constitute approximately 1.0 kb of sequence, were amplified by PCR and analyzed. HIV-1 variants from each region were resolved and excised from the gel; this was followed by direct sequencing of different viral variants. In 9 infected patients, a limited number of dominant sequence variants could be seen in the three regions, together with a faint background of minor variants. The use of DGGE makes it possible to obtain a direct estimate of overall HIV-1 sequence diversity within patient samples without an intermediate DNA cloning step.
Restriction-site PCR: a direct method of unknown sequence retrieval adjacent to a known locus by using universal primers.
Authors: Sarkar G , Turner R T , Bolander M E
Published: June 2007

Fast acquisition of unknown nucleotide sequences around a known sequence has important implication in molecular biology, especially in genome mapping. We have developed a method, termed restriction site polymerase chain reaction (RS-PCR), that utilizes specially designed primers that recognize, anneal, and sustain PCR. These primers, termed restriction site oligonucleotides (oligonucleotide primers specific for a given restriction enzyme recognition sequence or RSOs), could be generated corresponding to any restriction enzyme irrespective of the length of the recognition site and used as PCR primers corresponding to the unknown region of a DNA segment. In this method a first round of PCR is carried out in different tubes with a set of RSOs and a primer specific to the known region. A second round of PCR is then performed on the products of the first PCR with the same RSOs and another specific primer internal to the first one. Subsequently, the products of the last round of PCR are transcribed with an appropriate RNA polymerase and sequenced with a reverse transcriptase with an end-labeled specific primer internal to the second specific PCR primer. To demonstrate the applicability of RS-PCR in retrieving unknown sequences around a known sequence, we have used a set of four RSOs and three specific primers representing the known sequence and have successfully obtained hitherto unknown factor IX sequences (12 of 12 times) from three species starting from genomic DNA. The sequences obtained indicate the presence of a conserved stretch of 20 nucleotides in the 3' noncoding region of the factor IX gene.(ABSTRACT TRUNCATED AT 250 WORDS)
A PCR-based method for the analysis of human CD44 splice products.
Authors: van Weering D H , Baas P D , Bos J L
Published: June 2007

CD44 is a transmembrane glycoprotein involved in the interaction between cells and extracellular matrix. Several variant forms of CD44 exist, which differ from each other in the composition of both the intra- and extracellular domain of the protein. Post-translational modification and alternative RNA processing are responsible for this variation. Recently, it was found that certain variant CD44 proteins, containing extra sequences in the extracellular domain of the protein, are involved in metastatic spread of tumor cells. Variant CD44 proteins are also involved in immunological functions of T and B cells. A large variety of alternatively spliced CD44 mRNAs can be expressed by cells. We have developed a method for the analysis of CD44 mRNAs present in the cell. This reverse transcription-polymerase chain reaction (RT-PCR)-based method can be used to analyze the exon composition of each major CD44 mRNA species present in the cell. In this study we describe the analysis of CD44 mRNAs isolated from six different human cell lines.
Determination of 5' ends of specific mRNAs by DNA ligase-dependent amplification.
Authors: Bertling W M , Beier F , Reichenberger E
Published: June 2007

We established a novel way to clone 5' ends of unknown length and sequence of individual cDNAs. T4 DNA ligase is employed to ligate an annealed duplex of complementary primers, one of them with a 4-nucleotide-long randomized overlap, to first-strand cDNA, generating a new 5' end. Subsequent PCR with a down-stream primer and a primer with specificity for this new 5' end leads to products that can easily be cloned and sequenced. Considerations for the choice of primers for ligation and amplification are given. We have used this method to determine the 5' sequences of three independent mRNAs: the human collagen type-X gene, the chicken anchorin CII gene, and the human cytidine deaminase gene. We will discuss this method in comparison with other methods published for the amplification of unknown 5' ends of mRNA species.
A second locus for hereditary hemorrhagic telangiectasia maps to chromosome 12.
Authors: Johnson D W , Berg J N , Gallione C J , McAllister K A , Warner J P , Helmbold E A , Markel D S , Jackson C E , Porteous M E , Marchuk D A
Published: June 2007

Hereditary hemorrhagic telangiectasia (HHT) or Osler-Rendu-Weber (ORW) disease is an autosomal dominant vascular dysplasia. Initial linkage studies identified an ORW gene localized to 9q33-q34 but with some families clearly excluding this region. A probable correlation in clinical phenotype between the 9q3-linked families and unlinked families was described with a significantly lower incidence of pulmonary arteriovenous malformations observed in the unlinked families. In this study we examined four unrelated ORW families for which linkage to chromosome 9q33-q34 has been previously excluded. Linkage was established for all four families to markers on chromosome 12, with a combined maximum lod score of 10.77 (theta = 0.04) with D12S339. Mapping of crossovers using haplotype analysis indicated that the candidate region lies in an 11-CM interval between D12S345 and D12S339, in the pericentromeric region of chromosome 12. A map location for a second ORW locus is thus established that exhibits a significantly reduced incidence of pulmonary involvement.
Quantitative trait loci that modify the severity of spotting in piebald mice.
Authors: Pavan W J , Mac S , Cheng M , Tilghman S M
Published: June 2007

Mice homozygous for the recessive mutation piebald (s) exhibit a white-spotted coat caused by the defective development of neural crest-derived melanocytes. The severity of white spotting varies greatly, depending on the genetic background on which s is expressed. A backcross between two inbred strains of s/s mice that exhibit large differences in the degree of spotting was used to identify six genetic modifiers of piebald spotting on chromosomes 2, 5, 7, 8, 10, and 13. The loci differed in their spatial contribution to spotting on the dorsal versus ventral surfaces of mice; nonadditive interactions were observed between loci on chromosomes 2 and 5. This study underscores the power of using genetic analyses to identify and analyze loci involved in modifying the severity of phenotypic traits in mice.
Mouse galactokinase: isolation, characterization, and location on chromosome 11.
Authors: Ai Y , Jenkins N A , Copeland N G , Gilbert D H , Bergsma D J , Stambolian D
Published: June 2007

Elevated galactose levels can be caused by several enzyme defects, one of which is galactokinase. Galactokinase deficiency cause congenital cataracts during infancy and presenile cataracts in the adult population. We have isolated the mouse cDNA for galactokinase, which shares extensive amino acid sequence homology, 88% identity, with a recently cloned human galactokinase. It is expressed in all tissues examined. In an interspecific backcross analysis galactokinase maps to the distal region of mouse chromosome 11, a region that is homologous to human chromosome 17q22-25. The availability of the mouse gene provides an opportunity to make a knockout model for galactokinase deficiency.
Generation of entire human papillomavirus genomes by long PCR: frequency of errors produced during amplification.
Authors: Stewart A C , Gravitt P E , Cheng S , Wheeler C M
Published: June 2007

Recently, several improvements of traditional PCR techniques have facilitated the amplification of significantly longer DNA target sequences. Here we report an improved method for amplification of entire human papillomavirus (HPV) genomes. Using rTth DNA polymerase, XL (Perkin-Elmer, Foster City CA), and the accompanying XL PCR buffer system, we have successfully amplified 8-kb genomes from approximately 10 copies of input reference strain HPV16 DNA. This long PCR (LPCR) method was subsequently used to amplify the entire HPV16 genome from clinical specimens. The fidelity with which the rTth DNA polymerase XL amplified target sequences under our chosen amplification conditions was estimated by partial sequencing of cloned LPCR products generated from cloned reference strain HPV16 genomes. A region spanning the HPV16 E6, E7, and part of the E1 open reading frames (ORFs) was sequenced in 29 clones. A total of 33 nucleotide substitutions were observed in the 23.5 kb sequenced. This corresponds to an error frequency of approximately one error per 700 bases. Finally, LPCR methods were used to amplify entire, novel HPV genomes from clinical specimens. LPCR primer pairs were designed for amplification of seven potentially novel HPV types. Amplicons of approximately 8 kb were generated from five of the seven HPV types targeted. One of the LPCR-generated novel genomes, CP141, was subsequently cloned and a partial sequence was determined.
Heteroplasmy in the control region of human mitochondrial DNA.
Authors: Comas D , Pääbo S , Bertranpetit J
Published: June 2007

The sequencing of a 360-nucleotide segment of the mt DNA control region in a population survey revealed the presence of one individual having heteroplasmy in positions 16,293 (A and G) and 16,311 (C and T). This case indicates that heteroplasmy in humans may not be as rare as generally believed.
Evidence of a predisposing locus to bipolar disorder on Xq24-q27.1 in an extended Finnish pedigree.
Authors: Pekkarinen P , Terwilliger J , Bredbacka P E , Lönnqvist J , Peltonen L
Published: June 2007

An X-chromosomal predisposing locus to manic-depressive illness has been suggested since 1969 on the basis of the cosegregation of this trait in some families with phenotypic markers, such as color blindness, the glucose-6-phosphate dehydrogenase deficiency, and the coagulation factor IX deficiency. However, the conclusive evidence and the exact location of the putative X-chromosomal locus have remained controversial. We report here a linkage between DNA markers near the coagulation factor IX gene and bipolar disorder in an extended pedigree rising from the genetically isolated population of Finland. A distinct chromosomal haplotype covering a 20-cM region on Xq24-q27.1 could be demonstrated to segregate with bipolar disorder. These findings should encourage research groups to study extended family materials with Xq24-q27.1 markers to finally resolve the question of the X-chromosomal linkage of bipolar disorder.
Quantification of cytokine mRNA expression by RT-PCR and electrochemiluminescence.
Authors: Vandevyver C , Motmans K , Raus J
Published: June 2007

Variable gene expression constitutes a major mechanism for controlling cell development and cell function. To investigate these changed mRNA levels, a sensitive and quantitative assay is required. We describe a quick and easy method to quantify specific mRNAs by a combination of PCR and an electrochemiluminescent (ECL) detection of the amplified products. Total cellular RNA is reverse-transcribed and amplified with a biotinylated forward primer and a Tris (2,2'-bipyridine) ruthenium (II) (TBR)-labeled reverse primer. The amplification product is captured on streptavidin-coated paramagnetic beads and quantified by ECL detection using the QPCR system 5000. The results can be converted to quantitative values with an external standard curve. This method permits accurate and reliable quantitation of cytokine mRNA expression.
A method for accurate determination of terminal sequences of viral genomic RNA.
Authors: Weng Z , Xiong Z
Published: June 2007

A combination of ligation-anchored PCR and anchored cDNA cloning techniques were used to clone the termini of the saguaro cactus virus (SCV) RNA genome. The terminal sequences of the viral genome were subsequently determined from the clones. The 5' terminus was cloned by ligation-anchored PCR, whereas the 3' terminus was obtained by a technique we term anchored cDNA cloning. In anchored cDNA cloning, an anchor oligonucleotide was prepared by phosphorylation at the 5' end, followed by addition of a dideoxynucleotide at the 3' end to block the free hydroxyl group. The 5' end of the anchor was subsequently ligated to the 3' end of SCV RNA. The anchor-ligated, chimerical viral RNA was then reverse-transcribed into cDNA using a primer complementary to the anchor. The cDNA containing the complete 3'-terminal sequence was converted into ds-cDNA, cloned, and sequenced. Two restriction sites, one within the viral sequence and one within the primer sequence, were used to facilitate cloning. The combination of these techniques proved to be an easy and accurate way to determine the terminal sequences of SCV RNA genome and should be applicable to any other RNA molecules with unknown terminal sequences.
Molecular cloning and RARE cleavage mapping of human 2p, 6q, 8q, 12q, and 18q telomeres.
Authors: Macina R A , Morii K , Hu X L , Negorev D G , Spais C , Ruthig L A , Riethman H C
Published: June 2007

Large terminal fragments of human chromosomes 2p, 6p, 8q, 12q, and 18q were cloned using yeast artificial chromosomes (YACs). RecA-assisted restriction endonuclease (RARE) cleavage analysis of genomic DNA samples from II unrelated individuals using YAC-derived probes confirmed the telomeric localizations of the half-YACs studied. The cloned fragments provide telomeric closure of maps for the respective chromosome arms and will supply the reagents needed for analyzing and sequencing these distal subtelomeric regions.
Reduction of mispriming in amplification reactions with restricted PCR.
Authors: Puskás L G , Bottka S
Published: June 2007

A simple method is described for reducing nonspecific background, which is caused by mispriming during PCR. Besides the standard pair of primers, 3'-dideoxy-terminated competitor oligonucleotides were added to the amplification. Sequences to those of the primers which had identical base. In this way enhanced specificity was achieved. The competitor oligonucleotides may act by masking possible sites of nonspecific primer-template interaction, thus excluding undesired chain extensions. This technique is generally applicable when highly degenerate primers are used and therefore expands the potential of "restricted" PCR.
Generation of a high-resolution genetic map and a YAC contig of the Lurcher locus on mouse chromosome 6.
Authors: Zuo J , De Jager P L , Norman D J , Heintz N
Published: June 2007

Lurcher (Lc) is a semidominant mouse mutant that displays progressive neurodegeneration during perinatal development. This genetic lesion results in apoptotic neuronal death in a dosage dependent and cell autonomous manner in specific neurons during their terminal differentiation. To understand the molecular basis of the Lc mutation, we have adopted a positional cloning approach based on its location on mouse chromosome 6. To define the Lc locus, we have extended our previous analysis of an intersubspecific backcross between Mus m. castaneus and B6CBACa-Aw-j/A-Lc consisting of 504 animals (Norman et al. 1991). In addition, 580 animals of a generic backcross between Mus spretus and C57BL/6 (The European Collaborative Interspecific Backcross) were utilized for the fine genetic mapping of the Lc locus. Using three RFLP markers and nine microsatellite markers in the vicinity of the Lc locus, we determined the order and relative genetic distances of these markers at a resolution of 0.1 cM. The Lc mutation was mapped between two flanking markers, D6Mit121 and D6Mit175, separated by a genetic distance of 0.5 cM. We then initiated the cloning of the genomic region surrounding these two markers by screening a YAC library and characterizing YAC end sequences for further screening. This effort has resulted in the construction of a YAC contig consisting of 14 YACs and spanning a 3-Mb region. Markers isolated from these YACs were used to further define the Lc locus, resulting in a physical map that places the Lc gene within an estimated 300-kb interval. This set of YACs and markers will serve as DNA sources for the identification of the Lc gene.
An infrared fluorescent dATP for labeling DNA.
Authors: Steffens D L , Jang G Y , Sutter S L , Brumbaugh J A , Middendorf L R , Mühlegger K , Mardis E R , Weinstock L A , Wilson R K
Published: June 2007

Near-infrared fluorescence provides a nonradioactive method of detection with high sensitivity and low background. An infrared fluorophore has been attached covalently to the nucleotide deoxyadenosine triphosphate (dATP) to provide a reagent for enzymatic labeling of various types of DNA molecules and for facilitating their detection with an automated DNA sequencing and analysis system. DNA sequencing reaction products can be labeled internally by performing limited polymerization utilizing infrared-labeled dATP (IR-dATP) as the sole source of adenine deoxynucleotide prior to a dideoxy-specific termination reaction. PCR products can be labeled fluorescently by the addition of limited quantities of IR-dATP to the amplification reaction. This latter strategy has been utilized for detection of short tandem repeat polymorphisms (STRPs) which are useful for gene mapping, genetic diagnostics, forensic analysis, and paternity testing. Restriction fragments can be labeled also by fill-in reactions of appropriate 5' overhangs. Diminutive amounts of such fluorescently labeled DNA molecules can be visualized rapidly and conveniently using infrared detection technology.
RNA-primed PCR.
Authors: Shibata H , Tahira T , Hayashi K
Published: June 2007

We show that RNA can serve as a primer in PCR. Use of rTth DNA polymerase is essential because it has strong reverse transcriptase activity. RNA primers can be obtained by in vitro transcription and are less costly than DNA primers, which are chemically synthesized. RNA-primed PCR also opens the possibility that a specific amplification reaction can be achieved in the absence of knowledge of the target nucleotide sequence.
A high-fidelity physical map of human chromosome 21q in yeast artificial chromosomes.
Authors: Korenberg J R , Chen X N , Mitchell S , Fannin S , Gerwehr S , Cohen D , Chumakov I
Published: June 2007

Understanding of the human genome has been advanced significantly by the development of large DNA fragment libraries. To create a map of chromosome 21q that integrates the physical, cytogenetic, and linkage maps, we have characterized a subset of 127 chromosome 21 yeast artificial chromosome (YAC) clones for size, by pulsed field gel electrophoresis, for chimerism and cytogenetic location, by fluorescence in situ hybridization (FISH), and for sequence-tagged sites (STS) content, by PCR. It was found that 54% generated unique map locations on chromosome 21, and 45% detected sites on other chromosomes, of which 33% likely represented true chimerism. Using a simple algorithm, the data from nonchimeric clones have been combined to generate a size-corrected minimal tiling pathway including 58 chromosome 21q YACs that represent approximately 33 Mb and include 9 gaps. To confirm the resulting order and relationship to the cytogenetic map, the breakpoints from 23 cell lines partially aneuploid for chromosome 21 have been analyzed by quantitative Southern blot dosage analysis and FISH with a subset of the markers. As one way of investigating the relationship of the genetic to the physical map, the genetic map was superimposed on the physical map using a subset of well-defined markers common to both. The results suggest potential hot spots for recombination and/or gaps in the physical map. This integrated map will facilitate the search for the genes responsible for the Down syndrome phenotypes and provide a better understanding of genome organization and chromosome structure.
Human repeat-mediated integration of selectable markers into somatic cell hybrids.
Authors: Watson J E , Slorach E M , Maule J , Lawson D , Porteous D J , Brookes A J
Published: June 2007

We describe a strategy to introduce preferentially the dominant selectable marker neoR into the human chromosome within a monochromosome hybrid cell line. Integration of a construct containing the marker is mediated by human-specific repeat elements that promote multilocus human-specific integration with a single targeting vector. We tested two classes of repeat elements: the Alu family of SINE repeats and the Line1 repeat family. We show that Alu sequences alone are insufficient to direct human-specific integration but when used in combination with a Line1 element, or when only Line1 elements are included, integration of the vector into the human component of a monochromosome somatic cell hybrid is favored. The vectors also carry sequences that facilitate mapping and selective cloning of the targeted region. This strategy provides a means to generate selectable human subchromosomal fragments that can be used for localization of genes through positional cloning and, more important, for the identification of functional units through DNA transfer.
A novel in vivo method to detect DNA sequence variation.
Authors: Faham M , Cox D R
Published: June 2007

Mismatch repair detection (MRD) is an in vivo method that uses a change in bacterial colony color to detect DNA sequence variation. DNA fragments to be screened for variation are cloned into two MRD plasmids, and bacteria are transformed with heteroduplexes of these constructs. The resulting colonies are blue in the absence of a mismatch and white in the presence of a mismatch. MRD is capable of detecting a single mismatch in a DNA fragment as large as 10 kb in size. In addition, MRD has the potential for analyzing many fragments simultaneously, offering a powerful method for high-throughput genotyping and mutation detection in a large genomic region.
Accurate and absolute quantitative measurement of gene expression by single-tube RT-PCR and HPLC.
Authors: Hayward-Lester A , Oefner P J , Sabatini S , Doris P A
Published: June 2007

We report a method that allows accurate, absolute quantification of gene expression in a single reverse transcriptase (RT)-PCR reaction. This method makes use of novel high-performance liquid chromatography (HPLC) technology to resolve and quantify the products of competitive, mutant RNA PCRs. The HPLC technique allows rapid, high resolution of reaction products. On-line UV detection eliminates the need for radiolabel or other tracers. The HPLC technique also demonstrates that these competition reactions readily generate heteroduplex products. The ability of HPLC to resolve and quantify heteroduplex products is fundamental to the accuracy of the technique. Accurate measurements of gene expression have been obtained over four orders of magnitude and experiments employing predetermined quantities of specific native RNA input have demonstrated the ability of the system to provide absolute estimates of gene expression. Large size differences between native and mutant RNA inputs affected reverse transcriptase (RT) efficiency, but not PCR amplification efficiency. However, the magnitude of the RT efficiency effect can be estimated, is reproducible, and can therefore be adjusted by a calculated correction factor. The RT efficiency difference can been eliminated by reduction in the magnitude of the sequence difference between native and mutant RNA so that no correction factor is required. The application of the technique to quantification of expression of the alpha 1 subunit of sodium, potassium-ATPase in microdissected nephron segments is demonstrated.
High-quality automated DNA sequencing primed with hexamer strings.
Authors: Lodhi M A , McCombie W R
Published: June 2007

The finishing phase of genome sequencing projects is expensive, in part, because of the cost of de novo synthesis of custom primers and the management burden associated with obtaining and using them for primer walking. One approach to reduce these high costs is the use of a presynthesized library of short oligonucleotides (8-10 bases) rather than long primers. The use of such a library eliminates the need for custom synthesis of oligonucleotides, providing the convenience of priming from any site by combining two to three short oligonucleotides to form a string with the required specificity. The first practical implementation of this strategy presented a robust protocol for using hexamer strings with radioisotopic labelling. Whereas versions of this technique have subsequently been implemented on fluorescent sequencers we felt that there was a need to develop and extensively test a protocol that consistently gave read lengths comparable to dye-terminator sequencing with longer primers. We have developed a new two-cycle fluorescent Sequenase terminator procedure for using hexamer strings. We tested this procedure using a set of 32 different 3 hexamer primer strings, each known to be functional to some degree in radioisotopic sequencing, on single-stranded M13mp18 template and ABI 373 DNA sequencers. The overall success rate of priming with these hexamer primer strings is 97% with the failure of only one string. In this case, the corresponding 18-mer primer also failed to produce usable sequence from M13mp18 template. The average read length from reactions successfully primed with the 31 different hexamer strings was 461 bases with > 99% base-calling accuracy. The current protocol is robust enough to be used in virtually any situation where primer walking on single-stranded templates is used. The success rate and read lengths make it universally applicable to the sequencing of single-stranded templates on automated sequencers. It is also amenable to automation.
Transcriptional map of the Treacher Collins candidate gene region.
Authors: Loftus S K , Dixon J , Koprivnikar K , Dixon M J , Wasmuth J J
Published: June 2007

Treacher Collins syndrome (TCOF1) is a dominant disorder of craniofacial development that has been linked previosuly to a region of chromosome 5q31.3-32. Identification of recombination events in affected individuals has reduced the candidate gene region to a 0.5-Mb area between the loci RPS14 (proximal) and ANX6 [distal]. A transcriptional map of this candidate gene region, generated by analysis of exon amplification clones, has identified the genomic location of four genes, heparan sulfate-N-sulfotransferase-N-deacetylase, glutathione peroxidase, as well as two novel, previously uncharacterized genes. Each of these genes, based on their location, must be considered candidates for TCOF1 locus.
Identification of 4370 expressed sequence tags from a 3'-end-specific cDNA library of human skeletal muscle by DNA sequencing and filter hybridization.
Authors: Lanfranchi G , Muraro T , Caldara F , Pacchioni B , Pallavicini A , Pandolfo D , Toppo S , Trevisan S , Scarso S , Valle G
Published: June 2007

A systematic study on the mRNA species expressed in the human skeletal muscle is presented in this paper. To carry on this study, a new method has been developed for the construction of unbiased cDNA libraries specially designed for the production of ESTs corresponding to the 3'-end portion of the mRNAs. The method has been applied to human skeletal muscle, where the analysis of the transcription profile is particularly difficult for the presence of several very abundant transcripts. To detect and quantify high-level mRNAs, the first 1054 ESTs were obtained from randomly selected clones. The 10 most abundant transcripts accounted for > 45% of the clones. Subsequently, these transcripts were identified by filter hybridization, thus making DNA sequencing more productive. Overall, 4370 clones were identified: 3372 by DNA sequencing and 998 by filter hybridization. The number of groups of sequences identifying individual transcripts was relatively low compared with other tissues, resulting in a total of 934 groups out of 4370 ESTs. Of these, 719 groups were represented by only one sequence.
Utilization of FISH in positional cloning: an example on 13q22.
Authors: Laan M , Isosomppi J , Klockars T , Peltonen L , Palotie A
Published: June 2007

In positional cloning the initial assignment of a gene to a specific chromosomal locus is followed by physical mapping of the critical region. The construction of a high-resolution physical map still involves considerable effort. However, new high-resolution fluorescence in situ hybridization (FISH) techniques have facilitated this process substantially. Here we summarize a strategy that combines a spectrum of FISH techniques [metaphase, interphase, mechanically stretched chromosomes (MSCs), and fiber-FISH on free chromatin] for the construction and characterization of a high-resolution physical map for a positional cloning project. The chromosomal region 13q22, containing the locus of the variant form of the neuronal ceroid lipofuscinosis (vLINCL, CLN5) disease, serves here as an example for this process. We used metaphase FISH to exclude positionally a candidate gene, to refine the locus to 13q22, and to analyze the possible chimerism of the YACs in the region. Both metaphase and interphase FISH techniques were applied to determine the low-resolution distances between the restricting markers. FISH using MSCs confirmed the centromeric-telomeric order of the clones and facilitated the estimation of the size of the gaps between the clones. Finally, fiber-FISH was found to be the method of choice for the construction of an accurate high-resolution map of the contig established over the restricted region. Thus, FISH techniques in combination with genetic mapping data enabled the refinement of the initial 4-cM region to a high-resolution map of only 400 kb in length. Here the FISH strategy replaced the need for many laborious traditional physical mapping methods, e.g., pulsed-field gel electrophoresis.
Transcriptional analysis of the candidate region for incontinentia pigmenti (IP2) in Xq28.
Authors: Rogner U C , Heiss N S , Kioschis P , Wiemann S , Korn B , Poustka A
Published: June 2007

The hereditary form of incontinentia pigmenti (IP2) is a rare disorder characterized by abnormalities of the tissues and organs derived from the ectoderm and neuroectoderm and has been linked to Xq28 distal to the factor VIII gene (F8C). Four YAC clones covering the 1.1-Mb candidate region at the telomere of Xq28 were subjected to direct cDNA selection and Alu long-range PCR. The products of both methods were subsequently used to isolate 154 cosmid clones that were assembled into five cosmid contigs. This first-generation cosmid map covered the region almost entirely and was used as a basis for constructing a transcript map that was in turn integrated with the physical YAC and cosmid maps. To isolate specifically coding sequences, exon trapping and cDNA selection methods were combined. Exon trapping was carried out on YAC Alu-PCR products, YAC Alu long-range PCR products, and on pools of cosmids. The region-specific enriched cDNA library was then screened by using the exon trap products as complex probes. To ensure a more complete analysis, the products from cDNA selection experiments were also used to screen conventional oligo(dT) primed cDNA libraries. Twenty overlapping cDNA contigs were assembled and computer analyses were performed to identify EST hits, open reading frames, protein motifs, and protein sequence homologies. Five of the cDNA contigs corresponded to known sequences such as the factor VIII, c6.1A, and c6.1B. genes, and both distal copies of the factor VIII intron 22 repeat sequence. Expression patterns of the 15 new cDNA contigs were analyzed by Northern blot and RT-PCR studies and these data were integrated with expression data obtained from known EST sequences. Although a more detailed analysis of this 1.1-Mb region with respect to the structure and function of the genes will only ultimately be possible by a global sequencing approach, an analysis of all novel transcripts as candidate genes for incontinentia pigmenti is already in progress.
Bovine natural resistance associated macrophage protein 1 (Nramp1) gene.
Authors: Feng J , Li Y , Hashad M , Schurr E , Gros P , Adams L G , Templeton J W
Published: June 2007

The Bcg/Ity/Lsh locus is a major gene controlling early phases of infection with intracellular parasites in mice. Natural resistance associated macrophage protein 1 (Nramp1) has been shown to be the Bcg gene in mice. Analysis of a bovine cDNA homolog of murine Nramp1, designated as bovine NRAMP1, predicted a 548-amino-acid protein with hydrophobic domains, an amino-terminal SH3-binding domain, and a conserved consensus transport motif. Northern blotting indicated that bovine NRAMP1 was expressed primarily in macrophages and tissues of the recticuloendothelial system. Bovine NRAMP1 was mapped to BTA 2 within syntenic loci conserved on HSA 2q and MMU 1.
Site-selected mutagenesis of the Drosophila second chromosome via plasmid rescue of lethal P-element insertions.
Authors: Guo Y , Gillan A , Török T , Kiss I , Dow J A , Kaiser K
Published: June 2007

This paper describes a fast and efficient approach to correlating cloned genes with mutant phenotypes in Drosophila. We make use of a large collection D. melanogaster lines with recessive lethal insertions of a P[lacW] transposon on their second chromosome. Within this collection there clearly must be many insertions corresponding to Drosophila genes that have been cloned and characterized, e.g., via homology with cloned mammalian genes, but for which mutant phenotypes have yet to be identified. We have made use of the fact that P[lacW] contains a plasmid replicon to establish a collection of rescued plasmids containing genomic DNA flanking the sites of transposon insertion. Plasmids representing a total of 1836 lines were independently rescued and pooled in batches of 10 and 100. Pools of 100 plasmids were screened by hybridization with cDNAs corresponding to cloned second chromosome loci. Hybridizing pools were then narrowed down to single plasmids by a process of subdivision and rehybridization, and corresponding mutant lines were obtained. The success rate was better than one in four. This rate would undoubtedly be improved by the use of genomic DNA probes.
Complete genomic sequence and analysis of 117 kb of human DNA containing the gene BRCA1.
Authors: Smith T M , Lee M K , Szabo C I , Jerome N , McEuen M , Taylor M , Hood L , King M C
Published: June 2007

Over 100 distinct disease-associated mutations have been identified in the breast-ovarian cancer susceptibility gene BRCA1. Loss of the wild-type allele in > 90% of tumors from patients with inherited BRCA1 mutations indicates tumor suppressive function. The low incidence of somatic mutations suggests that BRCA1 inactivation in sporadic tumors occurs by alternative mechanisms, such as interstitial chromosomal deletion or reduced transcription. To identify possible features of the BRCA1 genomic region that may contribute to chromosomal instability as well as potential transcriptional regulatory elements, a 117,143-bp DNA sequence encompassing BRCA1 was obtained by random sequencing of four cosmids identified from a human chromosome 17 specific library. The 24 exons of BRCA1 span an 81-kb region that has an unusually high density of Alu repetitive DNA (41.5%), but relatively low density (4.8%) of other repetitive sequences. BRCA1 intron lengths range in size from 403 bp to 9.2 kb and contain the intragenic microsatellite markers D17S1323, D17S1322, and D17S855, which localize to introns 12, 19, and 20, respectively. In addition to BRCA1, the contig contains two complete genes: Rho7, a member of the rho family of GTP binding proteins, and VAT1, an abundant membrane protein of cholinergic synaptic vesicles. Partial sequences of the 1A1-3B B-box protein pseudogene and IFP 35, an interferon induced leucine zipper protein, reside within the contig. An L21 ribosomal protein pseudogene is embedded in BRCA1 intron 13. The order of genes on the chromosome is: centromere-1FP 35-VAT1-Rho7-BRCA1-1A1-3B-telomere.
A nonsense mutation in the cathepsin K gene observed in a family with pycnodysostosis.
Authors: Johnson M R , Polymeropoulos M H , Vos H L , Ortiz de Luna R I , Francomano C A
Published: June 2007

Pycnodysostosis (MIM 265800) is a rare, autosomal recessive skeletal dysplasia characterized by short stature, wide cranial sutures, and increased bone density and fragility. Linkage analysis localized the disease gene to human chromosome 1q21, and subsequently the genetic interval was narrowed to between markers D1S2612 and D1S2345. Expressed sequence tagged markers corresponding to cathepsin K, a cysteine protease highly expressed in osteoclasts and thought to be important in bone resorption, were mapped previously in the candidate region. We have identified a cytosine to thymidine transition at nucleotide 862 (GenBank accession no. S79895) of the cathepsin K coding sequence in the DNA of an affected individual from a large, consanguinous Mexican family. This mutation results in an arginine to STOP alteration at amino acid 241, predicting premature termination of cathepsin K mRNA translation. All affected individuals in this family were homozygous for the mutation, suggesting that this alteration may lead to pycnodysostosis. Recognition of the role of cathepsin K in the etiology of pycnodysostosis should provide insights into the pathogenesis and treatment of other disorders of bone remodeling, including osteoporosis.
The exon structure of the mouse Sc1 gene is very similar to the mouse Sparc gene.
Authors: McKinnon P J , Kapsetaki M , Margolskee R F
Published: June 2007

Sc1 and Sparc are two extracellular proteins sharing similarity in their carboxyl terminus, with 63% identity over a 232-amino-acid region. We have cloned and mapped the genomic locus of mouse Sc1. The mouse Sc1 gene contains 11 exons spanning approximately 35 kb of DNA. The genomic structure (exon/intron boundaries) of Sc1 exons 6 to exon 11 is identical to those of the similar portion of the Sparc gene. This suggests that Sc1 and Sparc originated from a common ancestral gene. Using fluorescence in situ hybridization analysis, Sc1 was localized to band 5E4 of mouse chromosome 5.
Alu fossil relics--distribution and insertion polymorphism.
Authors: Arcot S S , Adamson A W , Lamerdin J E , Kanagy B , Deininger P L , Carrano A V , Batzer M A
Published: June 2007

Screening of a human genomic library with an oligonucleotide probe specific for one of the young subfamilies of Alu repeats (Ya5/8) resulted in the identification of several hundred positive clones. Thirty-three of these clones were analyzed in detail by DNA sequencing. Oligonucleotide primers complementary to the unique sequence regions flanking each Alu repeat were used in PCR-based assays to perform phylogenetic analyses, chromosomal localization, and insertion polymorphism analyses within different human population groups. All 33 Alu repeats were present only in humans and absent from orthologous positions in several nonhuman primate genomes. Seven Alu repeats were polymorphic for their presence/absence in three different human population groups, making them novel identical-by-descent markers for the analysis of human genetic diversity and evolution. Nucleotide sequence analysis of the polymorphic Alu repeats showed an extremely low nucleotide diversity compared with the subfamily consensus sequence with an average age of 1.63 million years old. The young Alu insertions do not appear to accumulate preferentially on any individual human chromosome.
Mapping the RP2 locus for X-linked retinitis pigmentosa on proximal Xp: a genetically defined 5-cM critical region and exclusion of candidate genes by physical mapping.
Authors: Thiselton D L , Hampson R M , Nayudu M , Van Maldergem L , Wolf M L , Saha B K , Bhattacharya S S , Hardcastle A J
Published: June 2007

Genetic linkage studies have implicated at least two loci for X-linked retinitis pigmentosa (XLRP) on proximal Xp. We now report a defined genetic localization for the RP2 locus to a 5-cM interval in Xp11.3-11.23. Haplotype analysis of polymorphic markers in recombinant individuals from two XLRP families has enabled us to identify DXS8083 and DXS6616 as the new distal and proximal flanking markers for RP2. Using STS-content and YAC end-clone mapping, an approximately 1.2 Mb YAC contig has been established encompassing the proximal RP2 boundary and extending from T1MP1 to DXS1240 in Xp11.23. Several ESTs have been positioned and ordered on this contig, one of which is novel to the region, identified by sequence data-base match to a physically mapped YAC insert terminal STS. Integration of the genetic and physical data has placed four retinally expressed genes proximal to DXS6616, and thereby excluded them from a causitive role in RP2. This work now provides a much needed focus for positional cloning approaches to isolation of the defective gene.
Lane tracking software for four-color fluorescence-based electrophoretic gel images.
Authors: Cooper M L , Maffitt D R , Parsons J D , Hillier L , States D J
Published: June 2007

Software to track sample lanes automatically in four-color, fluorescence-based, electrophoretic gel images has been developed for application in large-scale DNA sequencing projects. Lanes and lane boundaries are tracked by analyzing a first difference approximation to the gradient of a vertically integrated and processed "brightness" profile. Initially lanes are located in a region of the gel image selected for good horizontal lane spacing and signal strength. The software uses models of expected lane and interlane spacing and lateral lane behavior to maintain accurate tracking on imperfect gels. In areas where intensity-based tracking is difficult, interpixel column correlation is also used to locate and define lane features. Summary statistics and compressed-in-time images are generated for user evaluation of tracking performance. The software developed has been tested successfully on gel images with degradations including significant horizontal lane motion (curving) and image artifacts, and is now in full-scale use in our sequencing projects.
Discovering distinct genes represented in 29,570 clones from infant brain cDNA libraries by applying sequencing by hybridization methodology.
Authors: Milosavljevic A , Zeremski M , Strezoska Z , Grujic D , Dyanov H , Batus S , Salbego D , Paunesku T , Soares M B , Crkvenjakov R
Published: June 2007

To discover all distinct human genes and to determine their patterns of expression across different cell types, developmental stages, and physiological conditions, a procedure is needed for fast, mutual comparison of hundreds of thousands (and perhaps millions) of clones from cDNA libraries, as well as their comparison against data bases of sequenced DNA. In a pilot study, 29,570 clones in duplicate from both original and normalized, directional, infant brain cDNA libraries were hybridized with 107-215 heptamer oligonucleotide probes to obtain oligonucleotide sequence signatures (OSSs). The OSSs were compared and clustered based on mutual similarity into 16,741 clusters, each corresponding to a distinct cDNA. A number of distinct cDNAs were successfully recognized by matching their 107-probe OSSs against GenBank entries, indicating the possibility of sequence recognition with only a few hundred randomly chosen oligomers.
A graph theoretic approach to the analysis of DNA sequencing data.
Authors: Berno A J
Published: June 2007

The analysis of data from automated DNA sequencing instruments has been a limiting factor in the development of new sequencing technology. A new base-calling algorithm that is intended to be independent of any particular sequencing technology has been developed and shown to be effective with data from the Applied Biosystems 373 sequencing system. This algorithm makes use of a nonlinear deconvolution filter to detect likely oligomer events and a graph theoretic editing strategy to find the subset of those events that is most likely to correspond to the correct sequence. Metrics evaluating the quality and accuracy of the resulting sequence are also generated and have been shown to be predictive of measured error rates. Compared to the Applied Biosystems Analysis software, this algorithm generates 18% fewer insertion errors, 80% more deletion errors, and 4% fewer mismatches. The tradeoff between different types of errors can be controlled through a secondary editing step that inserts or deletes base calls depending on their associated confidence values.