Abstract: The present invention provides a stable and efficient method for stimulating RNAi-related gene silencing effects. The present invention also provides a fast, simple and specific method for generating amplified cDNA-aRNA hybrids whose quantity and quality are high enough to be used in specific gene silencing transfection. This improved RNA-polymerase cycling reaction (RNA-PCR) relies upon a thermocycling procedure of in-vitro transcription and reverse transcription to bring up the amount of DNA-RNA hybrids up to two thousand folds within one round of the reaction. The resulting cDNA-aRNA product is useful for silencing either endogenous or exogenous gene expression in transfected cells.

Abstract: The present invention provides a fast, simple and reliable polymerase thermocycling reaction procedure for the linear amplification of nucleic acid sequences from cellular RNAs or genomes or both. The principle of this polymerase thermocycling reaction method relies upon the thermal cycling steps of promoter-linked nucleic acid template synthesis and in-vitro transcriptional amplification to bring up the amount of desired nucleic acid sequences up to two thousand fold within one cycle of the above procedure. Neither RNase H activity nor alkaline degradation is used in order to protect the RNA sequences of resulting amplified products which are ready for further applications, such as genechip/microarray analysis, in-vitro translation reaction and induction of RNA interference. Without the preferential amplification drawback of PCR/RT-PCR methods, the accuracy and resolution of current molecular biology analysis and diagnosis can be significantly improved by the present invention.

Abstract: The present invention provides novel compositions and methods for suppressing the expression of a targeted gene using mRNA-cDNA duplexes. The invention further provides novel methods and compositions for generating amplified mRNA-cDNA hybrids, whose quantity is high enough to be used for the invention's gene silencing transfection. This improved RNA-polymerase chain reaction method uses thermocycling steps of promoter-linked double-stranded cDNA or RNA synthesis, in vitro transcription and then reverse transcription to amplify the amount of mRNA-cDNA hybrids up to two thousand folds within one round of the above procedure.

Abstract: The present invention provides a fast, simple and specific method for generating a complete full-length cDNA library from single cells. The first reverse transcription of intracellular mRNAs with an oligo(dT)n-promoter primer introduces a recognition site for following transcription of newly reverse-transcribed cDNAs. The poly-nucleotide tailing of above cDNAs in addition to aforementioned promoter region further forms binding templates for specific PCR amplification. After repeating the reverse transcription, transcription, reverse transcription and PCR procedure, we can multiply a single copy of mRNA to two billion folds by calculation based upon the comparison between the amount of a synthesized cDNA library and that of theoretically presumed mRNAs within a cell (0.1 pg). In conjunction with a cell fixation and permeabilization step, the complete full-length cDNA library can be directly generated from few single cells without mRNA degradation.

Abstract: The present invention provides a fast, simple and direct covalent bond formation between two strands of nucleotide sequences. Non-modified first strand nucleotide sequences are hybridized with second strand nucleotide sequences, of which certain specific base structure(s) is modified by chemical reagents in order to generate covalent bonding with the first strand. While the hybridization of these two strand nucleotide sequences generates double-stranded hybrid duplexes between their homologues, covalent bond formation occurs in the region of modified base-pairs. Since neither a polymerase chain restriction nor a restriction enzyme digestion can be performed with the covalently bonded hybrid duplexes, the present invention can be used to subtract common sequences during subtractive hybridization, to inhibit nonspecific contamination during subcloning and to increase binding stability of antisense probes during in situ hybridization as well as gene therapy.

Abstract: The present invention provides a simple, specific and nontoxic gene knock-out method by formation of covalently bonding between modified probes and targeted sequences. When covalently modified first strand probes are hybridized with a second strand of targeted gene transcripts, certain modified bases of said first strand will interact with natural bases of said second strand to form covalent bonds by which the translation of said second strand is inhibited. Because the hybridization of said two strands generates covalent base-pairing only between their complementary homology region(s), such specificity increases the targeting efficiency of a gene knock-out system.

Abstract: Excess amount of modified subtracter DNA from control cells is generated by carboxylating the base structures of its certain nucleotides with chemical agents in order to introduce covalent affinity between the modified subtracter and a non-modified tester DNA. Hybridization of the control subtracter and the experimental tester DNA is performed with a heat-melting and then cool-reassociation technique. While the desired different (heterologous) sequences remain in the form of hydrogen-binding, common (homologous) sequences of the hybridized DNA are covalently bonded to each other. Since the covalent bonding of the common sequences can not be broken during a polymerase chain reaction, resulting in no amplification of the common sequences but great amplification of the desired different sequences. The desired DNA sequences present after such covalent homologue subtraction and selective amplification represent those DNA sequences which only exist in the tester but not in the subtracter DNA library.

Abstract: The present invention provides a method for fast, simple, and reliable isolation of desired different sequences from two DNA libraries. Excess amount of nucleotide analog-containing DNA subtracter from control cells is generated by incorporating nucleotide analog with a template-dependent extension reaction to introduce susceptible-sites for subsequent enzymatic digestion. Hybridization of the control subtracter and experimental DNA is performed with a heat-melting and then cool-reassociation technique. The hybridized DNAs are subtracted with nucleotide analog-removing enzyme first, resulting in nicking or gapping all nucleotide analog-containing hybrid duplexes which are further digested by single-strand-specific nuclease. Desired DNA sequences from the experimental cells, but not the control ones stay intact throughout the digestion procedure and can be selectively amplified at the end.