An optimized PCR-based procedure for production of 13C/15N-labeled DNA

We have substantially improved a procedure that we previously described for producing 13C/15N-labeled DNA (Chen et al., FEBS Lett. 436, 372-376, 1998) to provide an economical and straightforward approach to the preparation of labeled DNA. The conditions for the PCR reactions have been optimized to permit the use of low concentrations of the costly labeled dNTPs (50 microM for each). In addition, a rapid and high-yield purification procedure has been developed that allows us to obtain a high yield of very pure labeled DNA. These modifications to our original procedure permit us to obtain 1.9 mg of an 18 bp DNA oligomer from 20 mg of dNTPs (ca. 10% yield from the starting dNTPs). This is sufficient material for the preparation of 0.4 mM sample in a volume of 400 microl. In summary, this procedure is a cost-effective, time-efficient procedure for the production of labeled DNA for NMR studies.     

Multiplex amplification enabled by selective circularization of large sets of genomic DNA fragments

We present a method to specifically select large sets of DNA sequences for parallel amplification by PCR using target-specific oligonucleotide constructs, so-called selectors. The selectors are oligonucleotide duplexes with single-stranded target-complementary end-sequences that are linked by a general sequence motif. In the selection process, a pool of selectors is combined with denatured restriction digested DNA. Each selector hybridizes to its respective target, forming individual circular complexes that are covalently closed by enzymatic ligation. Non-circularized fragments are removed by exonucleolysis, enriching for the selected fragments. The general sequence that is introduced into the circularized fragments allows them to be amplified in parallel using a universal primer pair. The procedure avoids amplification artifacts associated with conventional multiplex PCR where two primers are used for each target, thereby reducing the number of amplification reactions needed for investigating large sets of DNA sequences. We demonstrate the specificity, reproducibility and flexibility of this process by performing a 96-plex amplification of an arbitrary set of specific DNA sequences, followed by hybridization to a cDNA microarray. Eighty-nine percent of the selectors generated PCR products that hybridized to the expected positions on the array, while little or no amplification artifacts were observed.     

In vitro polyoma DNA synthesis: Involvement of RNA in discontinuous chain growth

Short fragments of DNA (5 S) isolated by denaturation from polyoma replicative intermediates pulse-labeled in vitro were shown to have RNA covalently attached by three criteria: (1) such fragments were slightly denser than bulk viral DNA. (2) They could be labeled directly with α-³²P-labeled ribotriphosphates. (3) Alkaline hydrolysis of fragments labeled with α-³²P-labeled deoxynucleoside triphosphates showed ³²P transfer to 3′ ribonucleoside monophosphates. Except for a preference of transfer from dC, the link showed little sequence specificity. The data are compatible with the notion that all short fragments in replicating viral DNA are initiated by an RNA primer. This RNA is maximally 30 bases long and is rather short-lived.     

Supported PCR: an efficient procedure to amplify sequences flanking a known DNA segment

We describe a novel modification of the polymerase chain reaction for efficient in vitro amplification of genomic DNA sequences flanking short stretches of known sequence. The technique utilizes a target enrichment step, based on the selective isolation of biotinylated fragments from the bulk of genomic DNA on streptavidin-containing support. Subsequently, following ligation with a second universal linker primer, the selected fragments can be amplified to amounts suitable for further molecular studies. The procedure has been applied to recover T-DNA flanking sequences in transgenic tomato plants which could subsequently be used to assign the positions of T-DNA to the molecular map of tomato. The method called supported PCR (sPCR) is a simple and efficient alternative to techniques used in the isolation of specific sequences flanking a known DNA segment.     

Site-specific DNA splicing: a general procedure for the creation of a restriction site at a predetermined position in a DNA sequence

A method is described for creating any of a wide array of restriction sites at a predetermined position in a known DNA sequence. The method utilizes the exonuclease activity of BAL 31 and a specially designed bifunctional oligodeoxynucleotide linker. The desired restriction site is generated when the linker is ligated to those BAL 31-digested DNA fragments which end with the target sequence. The proper ligation product is then identified by a highly specific hybridization procedure. The method is versatile and specific and is especially useful in the isolation of functional elements of a gene.     

Lac repressor–operator interaction: N-terminal peptide backbone 1H and 15N chemical shifts upon complex formation with DNA

When the lac repressor tetramer is bound to its DNA operator, methylation protection shows the nearly symmetric operator half-sites are contacted asymmetrically. This asymmetric binding results from the DNA sequence/structure. The reported structure of lac repressor N-terminal fragment and an 11 base-pair operator left half-site provides no information concerning the effect of asymmetric binding, from left operator half-site to right half-site, upon the polypeptide backbone. We isolated uniformly ¹⁵N labeled 56 amino acid wild-type (HP56WT) and 64 residue mutant [Pro3>Tyr3] (HP64tyr3) lac repressor N-terminal DNA binding fragments for ¹H/¹⁵N NMR studies with the left and right operators separately. Spectral coincidence of these longer fragments, indicating structural similarity with a protease derived 51 amino acid fragment for which the amide correlations are assigned, allows for assignment of the common amide resonances. For both HP56WT and HP64tyr3, spectral overlap of the amide correlation peaks reveals the polypeptide backbones of the uncomplexed polypeptides are structurally similar. Likewise the complexes of the peptides to the 11 base-pair lac left operator half-site are similar. On the other hand, complexes of HP56WT and the left compared to the right lac operator half-site show different residues of the polypeptide are affected by binding different half-sites of the operator. Thus, the DNA sequence/structure transmits asymmetry to the polypeptide backbone of the interacting protein.     

连接介导PCR及其在体内足迹研究中的应用

连接介导聚合酶链反应是以连接反应为基础的单侧PCR技术,首先在DNA片段的一端连接上一个公共连接子,而后在这个连接子与另一个DNA序列特异的引物间扩增.Vent聚合酶的使用,延伸产物捕获及连接子标记选择策略的采用,大大提高了连接介导聚合酶链反应的敏感性.这一技术的发明大大促进了体内足迹等研究的进行.     

Tandem PCR: a novel and efficient unit amplification model for the preparation of small DNA fragments

The present DNA marker preparation with PCR amplification, one primer pair for one target DNA fragment, was very tedious and labor intensive. To develop a simple and efficient system for the preparation of small DNA fragments, a novel PCR amplification pattern was designed and tested, of which targeted small DNA fragments were amplified in groups as a unit with a specific synthetic vector as template DNA. The amplified units can be different dependent on the identities of the employed primers and give out variable combinations of small DNA fragments through complete or partial restrictive digestion with EcoRI. The novel pattern made the PCR amplification of small DNA fragments not only more efficient but also more economic than ever before. The tandem PCR pattern, as the most efficient and high throughput method for small DNA fragment preparation, has wide application for the production of various DNA markers and a good complementation to the larger DNA fragment preparation by complex synthetic vector fermentation.     

Quantitative and qualitative analysis of amplified DNA sequences by a competitive hybridization assay.

The presence of allelic sequence variations in DNA fragments can be easily detected by measuring the extent of DNA strand exchange between test double-stranded PCR products (target) and labeled standard double-stranded PCR products (probe). Under selected hybridization conditions, sequences identical to the probe decreased the formation of double-labeled hybrid, whereas differing sequences were not efficient enough to compete with the regeneration of the probe. A single base substitution in the target DNA increased the percentage of remaining double-labeled probe. A general procedure involving denaturation and hybridization in solution under different temperature conditions or using different probes enabled sequence identification. The degree of regeneration of double-labeled probe was determined using a bioluminescent assay. We evaluated the specificity of this method with two probes (108 and 131 bp) and several targets with different base substitutions.     

Directly labeled DNA probes using fluorescent nucleotides with different length linkers.

Directly labeled fluorescent DNA probes have been made by nick translation and PCR using dUTP attached to the fluorescent label, Cy3, with different length linkers. With preparation of probes by PCR we find that linker length affects the efficiency of incorporation of Cy3-dUTP, the yield of labeled probe, and the signal intensity of labeled probes hybridized to chromosome target sequences. For nick translation and PCR, both the level of incorporation and the hybridization fluorescence signal increased in parallel when the length of the linker arm is increased. Under optimal conditions, PCR yielded more densely labeled probes, however, the yield of PCR labeled probe decreased with greater linear density of labeling. By using a Cy3-modified dUTP with the longest linker under optimal conditions it was possible to label up to 28% of the possible substitution sites on the target DNA with reasonable yield by PCR and 18% by nick translation. A mechanism involving steric interactions between the polymerase, cyanine-labeled sites on template and extending chains and the modified dUTP substrate is proposed to explain the inverse correlation between the labeling efficiency and the yield of DNA probe synthesis by PCR.     

The Efficiency of DNA Labeling with Near-Infrared Fluorescent Dyes

The efficiency of DNA labeling was assessed for 2'-deoxyuridine 5'-triphosphate (dUTP) derivatives containing the Cy7 cyanine dye as a fluorophore. Two fluorescent Cy7-labeled dUTP analogs differed in the chemical structure of the linker between the fluorophore and nucleotide moieties. The efficiency of the polymerase chain reaction (PCR) and inhibition with modified nucleotides were estimated by real-time PCR. The efficiency of labeled nucleotide incorporation in PCR products was measured by quantitative electrophoresis. The efficiency of target DNA labeling was evaluated by binding the fluorescently labeled PCR products to a microarray of oligonucleotide probes immobilized in hydrogel drops (a biochip). The near-infrared hybridization signal was detected by digital luminescence microscopy. An increase in linker length was found to provide more efficient incorporation of the labeled nucleotide. Both of the compounds provided high sensitivity and high specificity of DNA testing via allele-specific hybridization on a biochip.

     

Multiplex amplification of ancient DNA

This method is designed to assemble long, continuous DNA sequences using minimal amounts of fragmented ancient DNA as template. This is achieved by a two-step approach. In the first step, multiple fragments are simultaneously amplified in a single multiplex reaction. Subsequently, each of the generated fragments is amplified individually using a single primer pair, in a standard simplex (monoplex) PCR. The ability to amplify multiple fragments simultaneously in the first step allows the generation of large amounts of sequence from rare template DNA, whereas the second nested step increases specificity and decreases amplification of contaminating DNA. In contrast to current protocols using many template-consuming simplex PCRs, the method described allows amplification of several kilobases of sequence in just one reaction. It thus combines optimal template usage with a high specificity and can be performed within a day.     

Understanding the sequence-dependence of DNA groove dimensions: implications for DNA interactions

Background

The B-DNA major and minor groove dimensions are crucial for DNA-protein interactions. It has long been thought that the groove dimensions depend on the DNA sequence, however this relationship has remained elusive. Here, our aim is to elucidate how the DNA sequence intrinsically shapes the grooves.

Methodology/Principal Findings

The present study is based on the analysis of datasets of free and protein-bound DNA crystal structures, and from a compilation of NMR ³¹P chemical shifts measured on free DNA in solution on a broad range of representative sequences. The ³¹P chemical shifts can be interpreted in terms of the BI↔BII backbone conformations and dynamics. The grooves width and depth of free and protein-bound DNA are found to be clearly related to the BI/BII backbone conformational states. The DNA propensity to undergo BI↔BII backbone transitions is highly sequence-dependent and can be quantified at the dinucleotide level. This dual relationship, between DNA sequence and backbone behavior on one hand, and backbone behavior and groove dimensions on the other hand, allows to decipher the link between DNA sequence and groove dimensions. It also firmly establishes that proteins take advantage of the intrinsic DNA groove properties.

Conclusions/Significance

The study provides a general framework explaining how the DNA sequence shapes the groove dimensions in free and protein-bound DNA, with far-reaching implications for DNA-protein indirect readout in both specific and non specific interactions.     

Nucleotide Sequence Analysis of DNA

There are three major obstacles to the analysis of the nucleotide sequence in a DNA molecule starting from a known location in the DNA molecule. First, it is difficult to obtain large quantities of homogeneous DNA. Second, even the smallest DNA molecules contain several thousand nucleotides which make sequence analysis prohibitive. Third, there are no highly base-specific DNAases available for degrading DNA for sequence analysis. We have overcome some of these obstacles; first, by incorporating highly labelled deoxynucleotides into DNA in vitro, small amounts of material can be used for sequence analysis. Second, the nucleotide sequence of DNA molecules can now be determined from the 5′-terminal. Thus, two dodecanucleotide sequences corresponding to the two cohesive ends of λ DNA have been determined¹ and a nona-decanucleotide sequence corresponding to one cohesive end of phage 186 DNA has been completed². So far, our approach is limited to starting the analysis from the 5′-ends of a DNA molecule. A more general approach is being developed for starting the analysis from other selected parts of a DNA molecule with the use of specifically designed primers.     

Enzymatic conversion of long DNA to small DNA fragments for the construction of short hairpin RNA expression libraries

Several techniques to enzymatically construct a short hairpin RNA (shRNA) expression library have been reported as tools for comprehensive genetic analyses by RNA interference. Our technique constructs an shRNA expression library from 25- to 35-bp DNA fragments by fragmenting given double-stranded DNA (dsDNA). We compared the following two procedures to efficiently prepare such small DNA fragments: one is the cleavage of dsDNA with deoxyribonuclease I (DNase I) in the presence of Mn²⁺ followed by blunting with T4 DNA polymerase, and the other is the introduction of nicks with DNase I in the presence of Mg²⁺ followed by blunting with the Klenow fragment. Consequently, the latter yielded the DNA fragments more efficiently. However, these DNA fragments were contaminated with fused DNA fragments that had originated from two regions of original dsDNA. Therefore, we used single-strand-specific exonucleases and succeeded in suppressing the production of such fused DNA fragments. Our technique allows the efficient conversion of given dsDNA to small DNA fragments.     

Fast high-resolution mapping of long fragments of genomic DNA based on single-molecule detection

Here we describe bacterial genotyping by direct linear analysis (DLA) single-molecule mapping. DLA involves preparation of restriction digest of genomic DNA labeled with a sequence-specific fluorescent probe and stained nonspecifically with intercalator. These restriction fragments are stretched one by one in a microfluidic device, and the distribution of probes on the fragments is determined by single-molecule measurement of probe fluorescence. Fluorescence of the DNA-bound intercalator provides information on the molecule length. Because the probes recognize short sequences, they encounter multiple cognate sites on 100- to 300-kb-long DNA fragments. The DLA maps are based on underlying DNA sequences of microorganisms; therefore, the maps are unique for each fragment. This allows fragments of similar lengths that cannot be resolved by standard DNA sizing techniques to be readily distinguished. DNA preparation, data collection, and analysis can be carried out in as little as 5 h when working with monocultures. We demonstrate the ability to discriminate between two pathogenic Escherichia coli strains, O157:H7 Sakai and uropathogenic 536, and we use DLA mapping to identify microorganisms in mixtures. We also introduce a second color probe to double the information used to distinguish molecules and increase the length range of mapped fragments.     

A protocol for the construction of microsatellite enriched genomic library

An improved protocol for constructing microsatellite-enriched libraries was developed. The procedure depends on digesting genomic DNA with a restriction enzyme that generates blunt-ends, and on ligating linkers that, when dimerized, create a restriction site for a different blunt-end producing restriction enzyme. Efficient ligation of linkers to the genomic DNA fragments is achieved by including restriction enzymes in the ligation reaction that eliminate unwanted ligation products. After ligation, the reaction mixture is subjected to subtractive hybridization without purification. DNA fragments containing microsatellites are captured by biotin-labeled oligonucleotide repeats and recovered using streptavidin-coated beads. The recovered fragments are amplified by PCR using the linker sequence as primer, and cloned directly into a plasmid vector. The linker has the sequence GTTT on the 5′ end, which promotes efficient adenylation of the 3′ ends of the PCR products. Consequently, the amplified fragments could be cloned into vectors without purification. This procedure enables efficient enrichment and cloning of microsatellite sequences, resulting in a library with a low level of redundancy.     

Modified inter-simple sequence repeat PCR protocol for use in conjunction with the LI-COR Gene ImagIR2 DNA analyzer

Inter-simple sequence repeat (inter-SSR) PCR was assessed for use in variety testing of chrysanthemum. This method was modified to allow detailed analysis of DNA profiles on a LI-COR Gene ImagIR2 DNA analyzer system. Protocols for unlabeled PCR were unsuccessful in producing labeled products when using infrared (IR) dye-labeled primers. Various modifications to the known protocols were investigated: (i) different ratios of labeled to unlabeled primer; (ii) various annealing temperatures; (iii) the use of an IR genotyping kit; (iv) end labeling; and (v) direct incorporation and cycle labeling. Successful amplification using labeled primers only occurred when two consecutive reactions were performed. The first PCR was performed using standard protocols for unlabeled reactions. The second PCR used a dilution of these reaction products as a template and 50% IR-labeled and unlabeled primer. The complete procedure leading to a high-resolution analysis of inter-SSR PCR products on a LI-COR system is reported for the first time. This system allows high-throughput fingerprinting with the potential for applications on a commercial scale.