Sequencing-independent method to generate oligonucleotide probes targeting a variable region in bacterial 16S rRNA by PCR with detachable primers

Oligonucleotide probes targeting the small-subunit rRNA are commonly used to detect and quantify bacteria in natural environments. We developed a PCR-based approach that allows synthesis of oligonucleotide probes targeting a variable region in the 16S rRNA without prior knowledge of the target sequence. Analysis of all 16S rRNA gene sequences in the Ribosomal Database Project database revealed two universal primer regions bracketing a variable, population-specific region. The probe synthesis is based on a two-step PCR amplification of this variable region in the 16S rRNA gene by using three universal bacterial primers. First, a double-stranded product is generated, which then serves as template in a linear amplification. After each of these steps, products are bound to magnetic beads and the primers are detached through hydrolysis of a ribonucleotide at the 3' end of the primers. This ultimately produces a single-stranded oligonucleotide of about 30 bases corresponding to the target. As probes, the oligonucleotides are highly specific and could discriminate between nucleic acids from closely and distantly related bacterial strains, including different species of VIBRIO: The method will facilitate rapid generation of oligonucleotide probes for large-scale hybridization assays such as screening of clone libraries or strain collections, ribotyping microarrays, and in situ hybridization. An additional advantage of the method is that fluorescently or radioactively labeled nucleotides can be incorporated during the second amplification, yielding intensely labeled probes.     

Uracil DNA glycosylase-mediated cloning of polymerase chain reaction-amplified DNA: application to genomic and cDNA cloning.

A simple and rapid method for cloning of amplification products directly from the polymerase chain reaction (PCR) has been developed. The method is based on the addition of a 12-base dUMP-containing sequence (CUACUACUACUA) to the 5' end of PCR primers. Incorporation of these primers during PCR results in the selective placement of dUMP residues into the 5' end of amplification products. Selective degradation of the dUMP residues in the PCR products with uracil DNA glycosylase (UDG) disrupts base pairing at the termini and generates 3' overhangs. Annealing of 3' protruding termini to vector DNA containing complementary 3' ends results in chimeric molecules which can be transformed, with high efficiency, without in vitro ligation. Directional cloning of PCR products has also been accomplished by incorporating different dU-containing sequences at the end of each PCR primer. Substitution of all dT residues in PCR primers with dU eliminates cloning of aberrant "primer dimer" products and enriches cloning of genuine PCR products. The method has been applied to cloning of inter-Alu DNA sequences from human placental DNA. Using a single primer, DNA sequences between appropriately oriented Alu sequences were amplified and cloned. Cloning of cDNA for the glyceraldehyde-3'-phosphate dehydrogenase gene from rat brain RNA was also demonstrated. The 3' end region of this gene was amplified by the 3' RACE method and the amplified DNA was cloned after UDG digestion. Characterization of cloned DNAs by sequence analysis showed accurate repair of the cloning junctions. The ligase-free cloning method with UDG should prove to be a widely applicable procedure for rapid cloning of PCR-amplified DNA.     

Annealing control primer system for improving specificity of PCR amplification

A novel primer designed to improve the specificity of PCR amplification, called the annealing control primer (ACP), comprises a tripartite structure with a polydeoxyinosine [poly(dI)] linker between the 3' end target core sequence and the 5' end nontarget universal sequence. We show that this ACP linker prevents annealing of the 5' end nontarget sequence to the template and facilitates primer hybridization at the 3' end to the target sequence at specific temperatures, resulting in a dramatic improvement of annealing specificity. The effect of this linker is demonstrated by the incorporation of ACP sequences as primers during the amplification of target nucleotide sequence and as hybridization probes in the genotyping of single nucleotide polymorphisms. This is the first report to show that a poly(dI) linker between two different sequences of ACP forms a bubble-like structure and disrupts or destabilizes DNA duplex formation at certain annealing temperatures.     

Signal amplification of padlock probes by rolling circle replication.

Circularizing oligonucleotide probes (padlock probes) have the potential to detect sets of gene sequences with high specificity and excellent selectivity for sequence variants, but sensitivity of detection has been limiting. By using a rolling circle replication (RCR) mechanism, circularized but not unreacted probes can yield a powerful signal amplification. We demonstrate here that in order for the reaction to proceed efficiently, the probes must be released from the topological link that forms with target molecules upon hybridization and ligation. If the target strand has a nearby free 3' end, then the probe-target hybrids can be displaced by the polymerase used for replication. The displaced probe can then slip off the targetstrand and a rolling circle amplification is initiated. Alternatively, the target sequence itself can prime an RCR after its non-base paired 3' end has been removed by exonucleolytic activity. We found the Phi29 DNA polymerase to be superior to the Klenow fragment in displacing the target DNA strand, and it maintained the polymerization reaction for at least 12 h, yielding an extension product that represents several thousand-fold the length of the padlock probe.     

Structure of products of the Moloney murine leukemia virus endogenous DNA polymerase reaction.

We have investigated the process by which the single-stranded RNA genome of Moloney murine leukemia virus is copied into DNA in vitro. DNA synthesis if initiated near the 5' end of the genome, and the elongation of the growing chain occurs by a jumping mechanism whereby the DNA synthesized at the 5' end of the genome is elongated along the 3' end. Unique DNA fragments synthesized beyond the 5' end of the genome in vitro have, at their 5' and 3' ends, copies of unique sequences from the 5' and 3' ends of the genome. These flank a copy of the 49- to 60-nucleotide terminally redundant sequence. These results indicate that the terminal redundancy serves as a "bridge" to allow a DNA molecule synthesized at the 5' end of the genome to serve as a primer for synthesis from the 3' end.     

Light-directed 5'-->3' synthesis of complex oligonucleotide microarrays

Light-directed synthesis of high-density microarrays is currently performed in the 3'-->5' direction due to constraints in existing synthesis chemistry. This results in the probes being unavailable for many common types of enzymatic modification. Arrays that are synthesized in the 5'-->3' direction could be utilized to perform parallel genotyping and resequencing directly on the array surface, dramatically increasing the throughput and reducing the cost relative to existing techniques. In this report we demonstrate the use of photoprotected phosphoramidite monomers for light-directed array synthesis in the 5'-->3' direction, using maskless array synthesis technology. These arrays have a dynamic range of >2.5 orders of magnitude, sensitivity below 1 pM and a coefficient of variance of <10% across the array surface. Arrays containing >150,000 probe sequences were hybridized to labeled mouse cRNA producing highly concordant data (average R(2) = 0.998). We have also shown that the 3' ends of array probes are available for sequence-specific primer extension and ligation reactions.     

Fingerprinting of bacterial genomes by amplification of DNA fragments surrounding rare restriction sites.

A method for generating limited representations of total bacterial DNA, without prior knowledge of the DNA sequence, has been developed. This method consists of three steps: digestion with two restriction enzymes, ligation of two oligonucleotide adapters corresponding to the restriction sites, and selective PCR amplification of the ligation products. The method relies on the use of two restriction enzymes with considerable differences in cleavage frequency of the investigated DNA and the ligation of two different oligonucleotides, each corresponding to one of the two cohesive ends of DNA fragments. Three subsets of DNA fragments are generated during digestion and subsequent ligation: terminated with the same oligonucleotide on both 5' ends of DNA fragments (two subsets) and terminated with two different oligonucleotides. Suppression PCR allows only the third subset of DNA fragments to be amplified exponentially. The method allows bacterial species strain differentiation on the basis of the different DNA band patterns obtained after electrophoresis in polyacrylamide gels stained with ethidium bromide and visualized in UV light.     

两种DNA探针杂交检测结核分支杆菌方法的研究

为改进结核杆菌DNA探针的特异性与实用性,研制了以生物素标记的两种对结核分支杆菌特异的DNA探针:一个5’端标记的20bp的寡核苷酸探针和一个采用PCR方法合成的188bp长链探针。两种探针分别与结核分支杆菌的全染色体DNA,以及基因组上IS6110序列的一段317bp的PCR扩增产物进行斑点杂交,以碱性磷酸酶(AP)催化的染色反应检测,测试了两个探针的敏感性和特异性。系统地比较研究了两种探针杂交检测条件:探针的浓度选择,杂交温度与洗膜温度的选择,以及杂交与洗膜温度对检测的敏感性与特异性的影响。寡核苷酸探针和188bp探针杂交检测纯化结核分支杆菌基因组DNA的敏感性分别为100ng与6ng,杂交检测PCR产物的敏感性分别是400pg与50pg。两探针的最佳杂交浓度均为40～160ng/ml,最佳杂交温度分别是42℃与68℃,最佳洗膜温度分别是60℃与60～68℃之间。两种探针均仅与结核分支杆菌及BCG有杂交信号,而与其它受试分支杆菌及非分支杆菌杂交结果都呈阴性。它们的特异性都很强,但188bp探针的敏感性约是寡核苷酸探针的7～16倍,而且188bp探针检测本底较低,是检测结核分支杆菌的较佳选择     

3' RACE walking along a large cDNA employing tiered suppression PCR

Large genes present particular cloning difficulties, especially when expressed at relatively low levels. We describe a novel method, termed 3' rapid amplification of cDNA ends (RACE) walking, for the rapid determination of unknown 3' flanking sequence of a large cDNA. The technique is a derivative of the anchored PCR 5' RACE procedure but includes a specific and limited second-strand cDNA synthesis and a tiered "panhandle" suppression of nonspecific products. The method generated 900 bp of new sequence for the large tammar wallaby ATRY gene in two easy steps, in which standard 3' RACE and PCR-based cDNA library walking proved unsuccessful. This robust approach represents a new tool for isolating unknown sequence under challenging cloning scenarios such as poor library representation, long coding regions, long 3' untranslated regions, and difficult template regions.     

A method for fabricating uni-dsDNA microarray chip for analyzing DNA-binding proteins

This paper describes an approach for preparing unimolecular double-stranded DNA (uni-dsDNA) microarray chip. In this method, the various target oligonucleotides containing a reverse complementary sequence at 5' end were firstly annealed to a same universal oligonucleotide with amino group at 5' end and immobilized on aldehyde-derivatized glass slide. An on-chip DNA polymerization reaction was then performed to elongate the universal oligonucleotides. After a denaturation and a followed intra-strand annealing, a hairpin structure was formed at the free 3' end of the immobilized oligonucleotides. Finally, another on-chip DNA polymerization was done to synthesize the uni-dsDNA microarray. Combining with a PCR amplification of chemically synthesized target oligonucleotides, this method was much cost-effective for production of the uni-dsDNA microarray. The uni-dsDNA microarray was verified applicable for detecting the presence and monitoring the DNA-binding activity of the sequence-specific DNA-binding proteins.     

Allelic discrimination by nick-translation PCR with fluorogenic probes.

Nick-translation PCR was performed with fluorogenic probes. Two probes were used: one complementary to a sequence containing the F508 codon of the normal human cystic fibrosis (CF) gene (wt DNA) and one complementary to a sequence containing the delta F508 three base pair deletion (mut DNA). Each probe contained a unique and spectrally resolvable fluorescent indicator dye at the 5' end and a common quencher dye attached to the seventh nucleotide from the 5' end. The F508/delta F508 site was located between the indicator and quencher. The probes were added at the start of a PCR containing mut DNA, wt DNA or heterozygous DNA and were degraded during thermal cycling. Although both probes were degraded, each probe generated fluorescence from its indicator dye only when the sequence between the indicator and quencher dyes was perfectly complementary to target. The identify of the target DNA could be determined from the post-PCR fluorescence emission spectrum.     

Cloning and expression of a novel lipase gene from Pseudomonas fluorescens B52

Here we describe an advanced polymerase chain reaction (PCR) technique, the compatible ends ligation inverse PCR (CELI-PCR) for chromosome walking. In CELI-PCR, several restriction enzymes, which produce compatible cohesive ends, were used to digest target DNA simultaneously or sequentially to produce DNA fragments of suitable size. DNA fragments were then easily circularized and PCR amplification could be carried out efficiently. The previous limitations of inverse PCR were overcome, such as unavailable restriction sites, poor template DNA circularization, and low amplification efficiency. Therefore, successive chromosome walking was performed successfully. Our work, isolating a 11,395-bp fragment from Gossypium hirsutum, was presented as an example to describe how CELI-PCR was carried out.     

The complete sequence of a unique RNA species synthesized by a DI particle of VSV.

The 2S RNA synthesized in vitro by the RNA polymerase of a defective interfering (DI) particle of vesicular stomatitis virus was labeled at its 3' terminus with 32P-cytidine 3', 5' bisphosphate and RNA ligase. Analysis of the labeled RNA showed that it was a family of RNAs of different length but all sharing the same 5' terminal sequence. The largest labeled RNA was purified by gel electrophoresis, and the sequence of 41 of its 46 nucleotides was determined by rapid RNA sequencing methods. The assignment of the remaining 5 nucleotides was made on the basis of an analysis of one of the smaller RNAs and published data. A new approach in RNA sequencing based on the identification of 3' terminal nucleotides of rna fragments originally present in the DI product or generated during the ligation reaction confirmed most of the sequence. The complete sequence of this 46 nucleotide long plus-sense RNA is: ppACGAAGACCACAAAACCAGAUAAAAAA UAAAAACCACAAGAGGGUC-OH. This RNA anneals to the RNA of the DI particle from which it was synthesized, indicating that its synthesis is template-specified. At least the first 17 and possibly all of the nucleotides are also complementary to sequences at the 3' end of two other VSV DI particles which were derived independently and whose genomes differ significantly in length. These data suggest a common 3' terminal sequence among all VSV DI particles which contain part of the Lgene region of the parental genome.     

Novel amplification of DNA in a hairpin structure: towards a radical elimination of PCR errors from amplified DNA

Errors introduced during PCR amplification set a selectivity limit for microsatellite analysis and molecular mutation detection methods since polymerase misincorporations invariably get confused with genuine mutations. Here we present hairpin-PCR, a new form of PCR that completely separates genuine mutations from polymerase misincorporations. Hairpin-PCR operates by converting a DNA sequence to a hairpin following ligation of oligonucleotide caps to DNA ends. We developed conditions that allow a DNA hairpin to be efficiently PCR-amplified so that, during DNA synthesis, the polymerase copies both DNA strands in a single pass. Consequently, when a misincorporation occurs it forms a mismatch following DNA amplification, and is distinguished from genuine mutations that remain fully matched. Error-free DNA can subsequently be isolated using one of many approaches, such as dHPLC or enzymatic depletion. We present feasibility for the main technical steps involved in this new strategy, conversion of a sequence to a hairpin that can be PCR-amplified from human genomic DNA, exponential amplification from picogram amounts, conversion of misincorporations to mismatches and separation of homoduplex from heteroduplex hairpins using dHPLC. The present hairpin-PCR opens up the possibility for a radical elimination of PCR errors from amplified DNA and a major improvement in mutation detection.