PCR libraries of ancient DNA using a generalized PCR method.

We describe a generalized PCR method that will amplify fragments of DNA without any knowledge of sequence using a single primer. Although we are presently using this method to amplify DNA fragments isolated from ancient preserved tissues, in effect, producing PCR libraries, it may prove to have other applications.     

Differential screening of phage-ab libraries by oligonucleotide microarray technology

A novel and efficient tagArray technology was developed that allows rapid identification of antibodies which bind to receptors with a specific expression profile, in the absence of biological information. This method is based on the cloning of a specific, short nucleotide sequence (tag) in the phagemid coding for each phage-displayed antibody fragment (phage-Ab) present in a library. In order to set up and validate the method we identified about 10,000 different phage-Abs binding to receptors expressed in their native form on the cell surface (10 k Membranome collection) and tagged each individual phage-Ab. The frequency of each phage-Ab in a given population can at this point be inferred by measuring the frequency of its associated tag sequence through standard DNA hybridization methods. Using tiny amounts of biological samples we identified phage-Abs binding to receptors preferentially expressed on primary tumor cells rather than on cells obtained from matched normal tissues. These antibodies inhibited cell proliferation in vitro and tumor development in vivo, thus representing therapeutic lead candidates.     

Rational screening of oligonucleotide combinatorial libraries for drug discovery.

Combinatorial strategies offer the potential to generate and screen extremely large numbers of compounds and to identify individual molecules with a desired binding specificity or pharmacological activity. We describe a combinatorial strategy for oligonucleotides in which the library is generated and screened without using enzymes. Freedom from enzymes enables the use of oligonucleotide analogues. This dramatically extends the scope of both the compounds and the targets that may be screened. We demonstrate the utility of the method by screening 2'-O-Methyl and phosphorothioate oligonucleotide analogue libraries. Compounds have been identified that bind to the activated H-ras mRNA and that have potent antiviral activity against the human herpes simplex virus.     

Comparison of complex DNA mixtures with generic oligonucleotide microchips.

The reproducibility of melting curves for repeated hybridizations of target DNA with generic oligonucleotide microchips is shown experimentally to depend on the character of matching between fragments of target DNA and immobilized oligonucleotides. The reproducibility of melting curves is higher for the perfect match duplexes and decreases as the number of mismatched pairs within duplexes increases. This effect was applied to the comparative analysis of complex DNA mixtures. We developed a scheme in which we can identify and discriminate between the probe oligonucleotides responsible for the distinctions between target DNA mixtures. A scheme is illustrated by comparing DNA mixtures corresponding to V-D-J genes connected with populations of mRNAs CDR3 TCR Vb (T-cell receptor beta complementarity determining region 3) from the thymus and pancreas of NOD mice. Our results demonstrate that generic microchips can be applied efficiently to the analysis of DNA mixtures.     

Construction of semi-randomized gene libraries with weighted oligonucleotide synthesis and PCR

Randomized gene libraries may be constructed and screened to find novel candidates with particular functions, and the applications can range widely, from protein engineering to selecting new microRNAs. Here we describe a technique to construct gene libraries using semi-randomized weighted oligonucleotide synthesis and end-to-end ligation. This method makes it possible to search the combinatorial space around a particular nucleotide sequence for a greater number of positions than is possible with fully randomized oligonucleotides. As an alternative to full cassette construction, library mutations can also be introduced through 'round-the-world PCR' approaches. Construction of a randomized gene cassette and cloning can typically be achieved in 2 weeks. Therefore, these are rapid and convenient methods to generate successive generations of libraries for iterative selection and optimization.     

Production of complex nucleic acid libraries using highly parallel in situ oligonucleotide synthesis

Generation of complex libraries of defined nucleic acid sequences can greatly aid the functional analysis of protein and gene function. Previously, such studies relied either on individually synthesized oligonucleotides or on cellular nucleic acids as the starting material. As each method has disadvantages, we have developed a rapid and cost-effective alternative for construction of small-fragment DNA libraries of defined sequences. This approach uses in situ microarray DNA synthesis for generation of complex oligonucleotide populations. These populations can be recovered and either used directly or immortalized by cloning. From a single microarray, a library containing thousands of unique sequences can be generated. As an example of the potential applications of this technology, we have tested the approach for the production of plasmids encoding short hairpin RNAs (shRNAs) targeting numerous human and mouse genes. We achieved high-fidelity clone retrieval with a uniform representation of intended library sequences.     

Integration of multiple PCR amplifications and DNA mutation analyses by using oligonucleotide microchip

We have developed a method for parallel independent on-chip amplification and the following sequence variation analysis of multiple DNA regions directly using microchip with an array of nanoliter gel pads containing specific sets of tethered primers. The method has three key features. First, DNA to be amplified is enriched at gel pads by its hybridization with immobilized primers. Second, different sets of specific primers are immobilized within various gel pads, and primers are detached within gel pads just before polymerase chain reaction to enhance the amplification. A gel pad may contain an additional permanently immobilized dormant primer that is activated to carry out the allele-specific primer extension reaction to detect mutations. Third, multiple polymerase chain reactions are confined within nanoliter gel pads covered and separated from each other with mineral oil. The method was applied to simultaneously identify several abundant drug-resistant mutations in three genes of Mycobacterium tuberculosis.     

Photophysical characterization of a FRET system using tailor-made DNA oligonucleotide sequences

We have carried out a detailed photophysical study of the FRET D/A pair consisting of a carbostyril donor and a Ru(II)bathophenanthroline complex acceptor in double-stranded synthetic DNA. Altogether 13 different double-stranded 30 base pair DNAs showing small incremental differences in the distances between donor and acceptor were synthesized. Using the fluorescence of the donor as well as of the acceptor, D/A separations were determined and compared to those derived from a well-established model for DNA distance calculations. The model calculations and anisotropy studies revealed that the donor can nearly be seen as a free rotator allowing the application of the established FRET data evaluation.     

Rapid competitive PCR using melting curve analysis for DNA quantification.

A rapid competitive PCR method was developed to quantify DNA on the LightCycler. It rests on the quantitative information contained in the melting curves obtained after amplification in the presence of SYBR Green I. Specific hybridization probes are not required. Heterologous internal standards sharing the same primer binding sites and having different melting temperatures to the natural PCR products were used as competitors. After a co-amplification of known amounts of the competitor with a DNA-containing sample, the target DNA can be quantified from the ratio of the melting peak areas of competitor and target products. The method was developed using 16S rDNA fragments from Streptococcus mutans and E. coli and tested against existing PCR-based DNA quantification procedures. While kinetic analysis of real-time PCR is well established for the quantification of pure nucleic acids, competitive PCR on the LightCycler based on an internal standardization was found to represent a rapid and sensitive alternative DNA quantification method for analysis of complex biological samples that may contain PCR inhibitors.     

Rapid detection of Salmonella subspecies I by PCR combined with non-radioactive hybridisation using covalently immobilised oligonucleotide on a microplate

Abstract A polymerase chain reaction (PCR)-based test was developed for the detection of Salmonella . One pair of oligonucleotide primers was designed to amplify a 93-bp fragment of a gene required for the invasion of HeLa cells by Salmonella ser Typhi strain Ty2. The amplified product was analysed by non-radioactive sandwich hybridisation in microtiter plates using two oligonucleotides. The capture oligonucleotide was covalently linked onto aminated wells of microtiter plates. The detection oligonucleotide was labelled with biotine. The hybrid molecules were detected by avidine conjugated with alkaline phosphatase and chromogenic substrate. The described combination of microplate sandwich hybridisation and PCR seems to be a suitable method for rapid detection of Salmonella subspecies I. It only requires a thermal cycler and a conventional microtiter reader, and can be readily done on a large scale.     

Rapid isolation of desired sequences from lone linker PCR amplified cDNA mixtures: Application to identification and recovery of expressed sequences in cloned genomic DNA

A simple and efficient method for the rapid isolation of specific sequences from PCR-amplified cDNA mixtures has been developed. cDNA mixtures obtained using lone linker PCR (Ko et al. 1990) appeared to be highly representative even though the starting material, 100 ng-2 g of total RNA, is much less than is required for making an ordinary cDNA library. With this method, cDNA mixtures were obtained from limited materials, including early mouse embryos and primordial germ cells. For selective enrichment of desired cDNAs, biotinylated probe was hybridized with the lone linker-linked cDNA in solution and the resulting probe-cDNA hybrid was captured by Streptavidin-coated magnetic beads. After appropriate washing, cDNA was released from the beads and subjected to amplification followed by cloning into a vector. Using genomic fragments isolated during chromosomal walking in the T/t complex of mouse Chromosome (Chr) 17, cDNAs encoding novel germ cell specific genes have been readily isolated by the above procedures. The method, termed random access retrieval of genetic information through PCR (RARGIP), will streamline the entire process from RNA to cDNA greatly. Its application potentials in various areas of molecular genetics will be discussed.