Locked nucleic acids (LNAs) are synthetic nucleic acid analogs that bind to complementary target molecules (DNA, RNA or LNA) with very high affinity. At the same time, this binding affinity is ...decreased substantially when the hybrids thus formed contain even a single mismatched base pair. We have exploited these properties of LNA probes to develop a new method for single nucleotide polymorphism genotyping. In this method, very short (hexamer or heptamer) LNA probes are labeled with either rhodamine or hexachlorofluorescein (HEX), and their hybridization to target DNAs is followed by measuring the fluorescence polarization (FP) of the dyes. The formation of perfectly complementary double‐stranded hybrids gives rise to significant FP increases, whereas the presence of single mismatches results in very small or no changes of this parameter. Multiplexing of the assay can be achieved by using differentially labeled wild‐type and mutant specific probes in the same solution. The method is homogeneous, and because of the use of extremely short LNA probes, the generation of a universal set of genotyping reagents is possible.
Heritable RNA interference (RNAi), triggered from stably expressed transgenes with an inverted repeat (IR) configuration, is an important tool for reverse genetic studies. Here we report on the ...development of stable RNAi in Anopheles stephensi mosquitoes, the major vector of human malaria in Asia. Trans genic mosquitoes stably expressing a RNAi transgene, designed to produce intron‐spliced double‐stranded RNA (dsRNA) targeting the green fluorescent protein EGFP gene, were crossed to an EGFP‐expressing target line. EGFP expression was dramatically reduced at both the protein and RNA levels. The levels of gene silencing depended upon the RNAi gene copy number and its site of integration. These results demonstrate that specific RNAi‐mediated knockdown of gene function can be achieved with high efficiency in Anopheles. This will be invaluable to systematically unravel the function of Anopheles genes determining the vectorial capacity of the malaria parasite.
Mammalian organs are typically comprised of several cell populations. Some (e.g. brain) are very heterogeneous, and this cellular complexity makes it difficult, if not impossible, to interpret ...expression profiles obtained with microarrays. Instruments, such as those manufactured by Leica or Arcturus, that permit laser capture microdissection of specific cells or cell groups from tissues were developed to solve this problem. To take full advantage of these instruments, however, one must be able to recognize cell populations of interest and, after they are harvested, to extract intact, unmodified RNA from them. Here we describe a novel, fast and simple method to fix and immunostain tissue sections that permits this to be done.
Protein combinatorial libraries provide new ways to probe the determinants of folding and to discover novel proteins. Such libraries are often constructed by expressing an ensemble of partially ...random gene sequences. Given the intractably large number of possible sequences, some limitation on diversity must be imposed. A non‐uniform distribution of nucleotides can be used to reduce the number of possible sequences and encode peptide sequences having a predetermined set of amino acid probabilities at each residue position, i.e., the amino acid sequence profile. Such profiles can be determined by inspection, multiple sequence alignment or physically‐based computational methods. Here we present a computational method that takes as input a desired sequence profile and calculates the individual nucleotide probabilities among partially random genes. The calculated gene library can be readily used in the context of standard DNA synthesis to generate a protein library with essentially the desired profile. The fidelity between the desired profile and the calculated one coded by these partially random genes is quantitatively evaluated using the linear correlation coefficient and a relative entropy, each of which provides a measure of profile agreement at each position of the sequence. On average, this method of identifying such codon frequencies performs as well or better than other methods with regard to fidelity to the original profile. Importantly, the method presented here provides much better yields of complete sequences that do not contain stop codons, a feature that is particularly important when all or large fractions of a gene are subject to combinatorial mutation.
PDF
