CONSTRUCTION OF Sesbania rostrata COSMID GENOMIC LIBRARY AND MOLECULAR CLONING OF LEGHEMOGLOBIN GENE

Using plant mini-Ti cosmid pEND4K, the cosmid genomic library of Sesbania rostratawas constructed. Sizes of plant DNA inserts in clones were 25- 33 kb. The restrictionmapping showed that different recombinant involved various types of plant DNA insert. 4clones containing leghemoglobin gene sequence of S. rostrata were obtained by in situ hy-bridization of colonies. The cloning of leghemoglobin gene sequence has been confirmedby plasmid DNA dot hybridization and Southern blot hybridization.     

Oligonucleotide fingerprinting with (CAC)5: nonradioactive in-gel hybridization and isolation of individual hypervariable loci

The first topic to be treated in this paper is the nonradioactive DNA fingerprinting by means of in-gel hybridization with digoxigenated (CAC)5. Besides the fact that time-consuming Southern blotting can be avoided, the dried agarose is an excellent matrix to produce background-free nonradioactive DNA fingerprints. There is no tendency of either the oligonucleotide probe or the antibody towards unspecific binding to the dried agarose. Prehybridization and blocking steps are therefore superfluous. Furthermore, we will discuss what effect the degree of crosslinking of the antibody-enzyme conjugates has. The second topic concerns the isolation and characterization of locus-specific probes from a human (CAC)5 fingerprint. The isolation and characterization of one variable probe, by screening complete genomic libraries, is described and discussed. This probe is compared to a hypervariable single-copy probe, isolated from a size-enriched genomic library. The sequence of the repeat flanking locus-specific probe is presented and a semi-specific, adaptor-mediated polymerase chain reaction was designed to amplify (CAC)n/(GTG)n flanking sequences.     

CHARACTERIZATION OF RAD4 GENE REQUIRED FOR ULTRAVIOLET-INDUCED EXCISION REPAIR OF Saccharomyces cerevisiae PROPAGATED IN Escherichia coli WITHOUT INACTIVATION

The previously isolated RAD4 gene designated as pPC1 from the genomic library of Saccharomyces cerevisiae (Yoon et al., 1985, Korean J. Genetics 7, 97-104) appeared to propagate in Escherichia coli and yet retained its complementing activity to rad4 mutants without inactivation. The subcloned RAD4 gene was found to be localized within a 2.5 kb DNA fragment flanking Bg1II and BamHI sites in the insert DNA, and was shown to have the same restriction map as a yeast chromosomal DNA, as determined by Southern hybridization. Tetrad analysis and pulse-field chromosome mapping have revealed that the cloned RAD4 gene can be mapped and integrated into the yeast chromosome V, the actual site of this gene. DNA-tRNA hybridization has shown that the isolated RAD4 gene did not contain a suppressor tRNA gene. These results have indicated that the pPC1 is a functional RAD4 gene playing a unique role involved in the nucleotide excision repair of yeast without any genetic change during amplification in E. coli.     

Oligonucleotide fingerprinting of arrayed genomic DNA sequences using LNA-modified hybridization probes

Recently, we established a robust method for the detection of hybridization events using a DNA microarray deposited on a nanoporous membrane. Here, in a follow-up study, we demonstrate the performance of this approach on a larger set of LNA-modified oligoprobes and genomic DNA sequences. Twenty-six different LNA-modified 7-mer oligoprobes were hybridized to a set of 66 randomly selected human genomic DNA clones spotted on a nanoporous membrane slide. Subsequently, assay sensitivity analysis was performed using receiver operating characteristic (ROC) curves. Comparison of LNA-modified heptamers and DNA heptamers revealed that the LNA modification clearly improved sensitivity and specificity of hybridization experiment. Clustering analysis was applied in order to test practical performance of hybridization experiments with LNA-modified oligoprobes in recognizing similarity of genomic DNA sequences. Comparing the results with the theoretical sequence clusters, we conclude that the application of LNA-modified oligoprobes allows for reliable clustering of DNA sequences which reflects the underlying sequence homology. Our results show that LNA-modified oligoprobes can be used effectively to unravel sequence similarity of DNA sequences and thus, to characterize the content of unknown DNA libraries.     

Characterization of an unusual variant mRNA of human lysosomal alpha-mannosidase

Lysosomal alpha-mannosidase (EC 3.2.1.24) is an exoglycosidase in the glycoprotein degradation pathway and is encoded by a 3.0 kb cDNA. A 2.3 kb cDNA from a minor species of HeLa cell mRNA was discovered by RT-PCR cloning. Southern blotting and PCR analysis of the HeLa cell genomic DNA showed that the 2.3 kb message was encoded by the lysosomal alpha-mannosidase gene. Sequence comparison of the cDNA with the corresponding genomic DNA indicated that the 2.3 kb message was generated by an unusual intra-exonic joining event.     

Rapid Electrochemical Assessment of Tumor Suppressor Gene Methylations in Raw Human Serum and Tumor Cells and Tissues Using Immunomagnetic Beads and Selective DNA Hybridization

We report a rapid and sensitive electrochemical strategy for the detection of gene‐specific 5‐methylcytosine DNA methylation. Magnetic beads (MBs) modified with an antibody for 5‐methylcytosines (5‐mC) are used for the capture of any 5‐mC methylated single‐stranded (ss)DNA sequence. A flanking region next to the 5‐mCs of the captured methylated ssDNA is recognized by hybridization with a synthetic biotinylated DNA sequence. Amperometric transduction at disposable screen‐printed carbon electrodes (SPCEs) is employed. The developed biosensor has a dynamic range from 3.9 to 500 pm and a limit of detection of 1.2 pm for the methylated synthetic sequence of the tumor suppressor gene O‐6‐methylguanine‐DNA methyltransferase (MGMT) promoter region. The method is applied in the 45‐min analysis of specific methylation in the MGMT promoter region directly in raw spiked human serum samples and in genomic DNA extracted from U‐87 glioblastoma cells and paraffin‐embedded brain tumor tissues without any amplification and pretreatment step.     

Use of peptide nucleic acid probes for detecting DNA single-base mutations by capillary electrophoresis

Peptide nucleic acid (PNA) oligomers can be used as probes in pre-gel hybridization experiments, as an alternative to Southern hybridization. In this technique, the PNA probe is hybridized to a cyanine-5 labeled DNA sample denatured at low ionic strength, and the mixture is directly injected for size separation into a capillary electrophoresis (CE) system equipped with laser-induced fluorescence (LIF) detector. The neutral backbone of PNA allows hybridization to occur at low ionic strength and assures an efficient CE separation of the PNA/DNA hybrids from both double-stranded and single-stranded DNA. We have used as a model system the cystic fibrosis R553X and R1162X single-base mutations and we have assessed the influence of various factors, such as temperature and denaturants concentration on DNA/PNA hybrid stability in order to achieve the high specificity required for a single base pair discrimination.     

Polyaniline based nucleic acid sensor

Twenty-bases long NH2-modified DNA and PNA probes specific to a pathogen (Mycobacterium tuberculosis) were covalently immobilized onto a polyaniline (PANI)/Au electrode to detect nucleic acid hybridization with complementary, one-base mismatch and noncomplementary targets within 30 s using Methylene Blue. The PNA-PANI/Au electrode exhibits improved specificity (1000 times) and detection limit (0.125 x 10(-18) M) as compared to that of the DNA-PANI/Au electrode (2.5 x 10(-18) M). These PNA-PANI/Au electrodes can be utilized for detection of hybridization with the complementary sequence in 5 min sonicated M. tuberculosis genomic DNA within 1 min of hybridization time. These DNA-PANI/Au and PNA-PANI/Au electrodes can be used 6-7 and 13-15 times, respectively.     

Hybridization efficiency of molecular beacons bound to gold nanowires: effect of surface coverage and target length

Surface-bound nucleic acid probes designed to adopt specific secondary structures are becoming increasingly important in a range of biosensing applications but remain less well characterized than traditional single-stranded probes, which are typically designed to avoid secondary structure. We report the hybridization efficiency for surface-immobilized hairpin DNA probes. Our probes are molecular beacons, carrying a 3' dye moiety and a 5' thiol for attachment to gold nanowires, which serve as both scaffolds for probe attachment and quenchers. Hybridization efficiency was dependent on probe surface coverage, reaching a maximum of ～90% at intermediate coverages of (1-2) × 10(12) probes/cm(2) and dropping to ≤20% at higher or lower coverages. Fluorescence intensity did not track with the number of target molecules bound, and was highest for high probe coverage despite the lower bound targets per square centimeter. Backfilling with short thiolated oligoethylene glycol spacers increased hybridization efficiency at low hairpin probe coverages (～(3-4) × 10(11) probes/cm(2)), but not at higher probe coverages (1 × 10(12)/cm(2)). We also evaluated the effect of target length by adding up to 50 nonhybridizing nucleotides to the 3' or 5' end of the complementary target sequence. Additional nucleotides on the 3' end of the complementary target sequence (i.e., the end near the nanowire surface) had a much greater impact on hybridization efficiency as compared to nucleotides added to the 5' end. This work provides guidance in designing sensors in which surface-bound probes designed to adopt secondary structures are used to detect target sequences from solution.     

电化学石英晶体微天平实时表征和定量检测短序列DNA

利用电化学石英晶体微天平(EQCM)这一灵敏的质量和电化学传感器测定特定序列DNA。应用自组装膜技术在压电石英晶振表面自组装一带羧基的α－硫辛酸单层膜,通过盐酸1-乙基-3-(3-二甲基氨基丙基)碳二亚胺(EDC)及N-羟基琥珀酰亚胺(NHS)共价固化寡聚核苷酸为探针,用于测定与其碱基序列互补的DNA。实验中EQCM实时监测了α-硫辛酸的自组装过程、探针固化过程及其与cDNA杂交过程。定量得出了探针固化量及cDNA杂交量。在酸性、中性和碱性条件下,分别对固化和杂交过程进行表征,实验发现探针固化及DNA杂交都受pH影响,本文对此现象进行了解释。同时,利用染料Hoechst33258的电化学活性,使其与双链DNA嵌合,通过测量Hoechst33258的电化学信息进一步验证了DNA杂交关键步骤。     

The application of magnetic bead hybridization for the recovery and STR amplification of degraded and inhibited forensic DNA

A common problem in the analysis of forensic DNA evidence is the presence of environmentally degraded and inhibited DNA. Such samples produce a variety of interpretational problems such as allele imbalance, allele dropout and sequence specific inhibition. In an attempt to develop methods to enhance the recovery of this type of evidence, magnetic bead hybridization has been applied to extract and preconcentrate DNA sequences containing short tandem repeat (STR) alleles of interest. In this work, genomic DNA was fragmented by heating, and sequences associated with STR alleles were selectively hybridized to allele-specific biotinylated probes. Each particular biotinylated probe-DNA complex was bound to streptavidin-coated magnetic beads using enabling enrichment of target DNA sequences. Experiments conducted using degraded DNA samples, as well as samples containing a large concentration of inhibitory substances, showed good specificity and recovery of missing alleles. Based on the favorable results obtained with these specific probes, this method should prove useful as a tool to improve the recovery of alleles from degraded and inhibited DNA samples.     

Hybridization and enzymatic extension of au nanoparticle-bound oligonucleotides

We have investigated the impact of steric effects on the hybridization and enzymatic extension of oligonucleotides bound to 12-nm colloidal Au particles. In these experiments, a nanoparticle-bound 12-mer sequence is hybridized either to its solution phase 12-mer complement or to an 88-mer template sequence. The particle-bound oligonucleotide serves as a primer for enzymatic extension reactions, in which covalent incorporation of nucleotides to form the complement of the template is achieved by the action of DNA polymerase. Primers were attached via-C(6)H(12)SH, -C(12)H(24)SH, and -TTACAATC(6)H(12)SH linkers attached at the 5' end. Primer coverage on the nanoparticles was varied by dilution with (5')HSC(6)H(12)AAA AAA(3'). Hybridization efficiencies were determined as a function of linker length, primer coverage, complement length (12-mer vs 88-mer), and primer:complement concentration ratio. In all cases, hybridization for the 88-mer was less efficient than for the 12-mer. Low primer surface coverage, greater particle-primer separation, and higher primer:complement ratios led to optimal hybridization. Hybridization efficiencies as high as 98% and 75% were observed for the 12-mer and 88-mer, respectively. Enzymatic extension of particle-bound primers was observed under all conditions tested; however, the efficiency of the reaction was strongly affected by linker length and primer coverage. Extension of primers attached by the longest linker was as efficient as the solution-phase reaction.     

Enzyme-catalyzed signal amplification for electrochemical DNA detection with a PNA-modified electrode

The signal amplification technique of peptide nucleic acid (PNA)-based electrochemical DNA sensor was developed in a label-free and one-step method utilizing enzymatic catalysis. Electrochemical detection of DNA hybridization on a PNA-modified electrode is based on the change of surface charge caused by the hybridization of negatively charged DNA molecules. The negatively charged mediator, ferrocenedicarboxylic acid, cannot diffuse to the DNA hybridized electrode surface due to the charge repulsion with the hybridized DNA molecule while it can easily approach the neutral PNA-modified electrode surface without the hybridization. By employing glucose oxidase catalysis on this PNA-based electrochemical system, the oxidized mediator could be immediately reduced leading to greatly increased electrochemical signals. Using the enzymatic strategy, we successfully demonstrated its clinical utility by detecting one of the mutation sequences of the breast cancer susceptibility gene BRCA1 at a sample concentration lower than 10(-9) M. Furthermore, a single base-mismatched sample could be also discriminated from a perfectly matched sample.     

Detection of fragmented genomic DNA by PCR-free piezoelectric sensing using a denaturation approach

Label-free and real-time DNA sequence detection in PCR-amplified DNA samples can now be achieved by different approaches. On the contrary, only few works have been reported dealing with direct sequence detection in nonamplified genomic DNA. Here, a piezoelectric biosensor for direct detection of sequences in nonamplified genomic DNA is described. The system relies on real-time and label-free detection of the hybridization reaction between an immobilized probe and the complementary sequence in solution. The DNA probe is immobilized on the sensing surface (10 MHz quartz crystals), while the complementary sequence is present in the genomic DNA, previously fragmented with restriction enzymes.     

Targeted next-generation sequencing by specific capture of multiple genomic loci using low-volume microfluidic DNA arrays

We report a flexible method for selective capture of sequence fragments from complex, eukaryotic genome libraries for next-generation sequencing based on hybridization to DNA microarrays. Using microfluidic array architecture and integrated hardware, the process is amenable to complete automation and does not introduce amplification steps into the standard library preparation workflow, thereby avoiding bias of sequence distribution and fragment lengths. We captured a discontiguous human genomic target region of 185 kb using a tiling design with 50mer probes. Analysis by high-throughput sequencing using an Illumina/Solexa 1G Genome Analyzer revealed 2150-fold enrichment with mean per base coverage between 4.6 and 107.5-fold for the individual target regions. This method represents a flexible and cost-effective approach for large-scale resequencing of complex genomes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Stephan Bau and Nadine Schracke contributed equally to this work.     

Chemiluminescent detection of DNA hybridization and single-nucleotide polymorphisms on a solid surface using target-primed rolling circle amplification

A new chemiluminescent (CL) method has been developed for the sensitive detection of DNA hybridization and single-nucleotide polymorphisms (SNPs) with target-primed rolling circle amplification (RCA). The capture oligonucleotide probe is firstly immobilized on a polystyrene well plate and then hybridized with the wild DNA target. A designed padlock probe is circularized after perfect hybridization to the DNA target. Then the RCA reaction can be initiated from the DNA target that acts as a primer and generates a long tandem single-strand of DNA with repeat sequences. In contrast, the mutant DNA target, which contains a mismatched base with the padlock probe, cannot initiate the RCA reaction and primes only a limited extension with the unligated padlock probe. Afterwards, a biotinylated oligonucleotide is used to hybridize with the RCA product in each repeat sequence and streptavidin-alkaline phosphatase (STV-AP) is employed to combine the anchored biotin. The DNA target is detected with the CL reaction of STV-AP and 3-(2'-spiroadamantane)-4-methoxy-4-(3'-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD). With the RCA-based method, the sensitivity of DNA detection can be increased by about two orders of magnitude compared with that of direct DNA hybridization. A DNA target as low as 3.6 pM can be detected. Wild-type DNA and the one-base mutant DNA can be differentiated with high selectivity through this RCA reaction.     

Use of vitamin B2 for fluorescence detection of thymidine-related single-nucleotide polymorphisms

In combination with abasic site (AP site)-containing DNAs, potential use of a biotic fluorescence compound, Vitamin B₂ (riboflavin), is demonstrated for the fluorescence detection of the thymine (T)-related single-nucleotide polymorphisms. Our method is based on construction of the AP site in DNA duplexes, which allows small ligands to bind to target nucleotides accompanied by fluorescence signaling: an AP site-containing probe DNA is hybridized with a target DNA so as to place the AP site toward a target nucleobase, by which hydrophobic microenvironments are provided for ligands to recognize target nucleotides through stacking and hydrogen-bonding interactions. In 10 mM sodium cacodylate buffer solutions (pH 7.0) containing 100 mM NaCl and 1.0 mM EDTA, Vitamin B₂ is found to selectively bind to T (K₁₁ = 1.8 × 10⁶ M⁻¹ at 5 °C) over other nucleobases, and this is accompanied by significant quenching of its fluorescence. While the sensing functions depend on the flanking sequences to the AP site, Vitamin B₂ is applicable to the detection of T/C (cytosine), T/G (guanine) and T/A (adenine) mutation sequences of the CYP2A6 gene, where the flanking nucleobases are guanines in both positions (-GXG-, X = AP site).     

DNA attachment chemistry at the flexible silicone elastomer surface: toward disposable microarrays

This paper describes the preparation and surface characterization of maleimide-activated silicone elastomer (PDMS(MCC)) followed by covalent functionalization using thiol-terminated DNA sequences (primary oligo). The stability of this attachment chemistry was demonstrated by the retention of the primary oligo through the process of hybridization with a labeled complementary DNA sequence. In these studies, the hybridized labeled DNA oligomers were detected using confocal fluorescence microscopy. We have employed a vapor deposition technique in which a plasma-treated silicone elastomer (PDMS(OH)) was exposed to vapors of 3-(aminopropyl)triethoxysilane (APTS) under vacuum, to yield the amine-functionalized silicone elastomer (PDMS(NH)(2)). PDMS(NH)(2) was further coupled with a heterofunctional cross-linker, sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate to obtain PDMS(MCC). The surface functionalities of the elastomers were characterized using contact angle measurements and X-ray photoelectron spectroscopy. Surface-modified silicone elastomers appear to be promising substrates for use as substrates for disposable microarrays.     

Multiplex polymerase chain reaction method for detection of bovine materials in foodstuffs

Rapid identification of bovine materials in animal foodstuffs is essential for effective control of a potential source of bovine spongiform encephalophathy. A convenient polymerase chain reaction (PCR)-based assay was developed for detection and identification of a bovine-specific genomic DNA sequence in foodstuffs. Simultaneously the assay assessed the DNA quality of the experiment system by amplification of a highly conserved eucaryotic DNA region of the 18-S ribosomal gene, helping to check the reliability of the test result. The amplified bovine-specific PCR product was a genomic DNA fragment of lactoferrin, a low copy gene that was different from a commonly used bovine-specific mitochondria sequence for identification of bovine materials. The specificity of this method was confirmed by the absence of detectable homologous PCR product using reference foodstuff samples that lacked bovine-derived meat and bonemeals, or genomic DNA samples from vertebrates whose offals are commonly included in animal feeds. This method could detect the presence of bovine material in foodstuffs when the samples contained > 0.02% bovine-derived meat and bone meal. Furthermore, it was not affected by prolonged heat treatment. The specificity, convenience, and sensitivity of this method suggest that it can be used for the routine detection of bovine-derived materials.