We have improved the "polymerase chain reaction" (PCR) to permit rapid analysis of any known mutation in genomic DNA. We demonstrate a system, ARMS (Amplification Refractory Mutation System), that allows genotyping solely by inspection of reaction mixtures after agarose gel electrophoresis. The system is simple, reliable and non-isotopic. It will clearly distinguish heterozygotes at a locus from homozygotes for either allele. The system requires neither restriction enzyme digestion, allele-specific oligonucleotides as conventionally applied, nor the sequence analysis of PCR products. The basis of the invention is that unexpectedly, oligonucleotides with a mismatched 3'-residue will not function as primers in the PCR under appropriate conditions. We have analysed DNA from patients with alpha 1-antitrypsin (AAT) deficiency, from carriers of the disease and from normal individuals. Our findings are in complete agreement with allele assignments derived by direct sequencing of PCR products.
The genomic RNA of the avian influenza A virus, fowl plague, was fractionated into eight species by electrophoresis in lyacrlamide-agarose gels containing 6 M urea. The separate 32P-labeled RNA species were characterized by digestion with RNase T, and fractionation of the resulting oligonucleotides by two-dimensional gel electrophoresis; this demonstrated that each species has a distinct nucleotide sequence. A tentative correlation of each genome RNA species with the virus protein that it encodes was made.A number of years ago, studies on the genetic and biological properties of influenza virus showed that the genetic material of the virus behaved as though it were composed of several individual units (1-3). More recently, physical studies of the genome RNA of influenza viruses, by sucrose density gradient centrifugation and by polyacrylamide gel electrophoresis, demonstrated that the RNA consists of a number of size classes with molecular weights between about 200,000 and 1,000,000, none of them large enough to represent the entire genetic material of the virus (4)(5)(6)(7)(8). It was also demonstrated by oligonucleotide mapping that three size classes of virus RNA contained distinct sequences, supporting the idea that fractions obtained by gel electrophoresis represented unique species rather than fragments of a larger RNA molecule (9). However, until now it has not been possible to separate the various RNA species completely, so that a complete description of influenza virus RNA in terms of the number of species, their relative representation, their molecular weights, and their nucleotide sequence relationships has not been possible. Despite this lack of resolution, the present, generally accepted view is that each RNA species contains the genetic information for one virus polypeptide (10). This independent nature of the virus genes is then held to account for the characteristic phenomena of high recombination frequencies in mixed infections (1, 3) and multiplicity reactivation of UV-irradiated virus (2) and is the basis for one theory concerning the origin of new, pandemic strains of influenza virus (11,12).We have found that a greatly improved fractionation of influenza virus RNA can be achieved by the use of polyacrylamide-agarose gels containing a high level of urea. In this paper we describe the separation of the virus RNA into eight species by this method. We also report our subsequent characterization of each RNA species by oligonucleotide mapping using twodimensional polyacrylamide gel electrophoresis. We conclude that the influenza virus genome is composed of eight RNA species containing distinct nucleotide sequences, and we attempt to correlate the genome RNA species with the virus proteins that they specify. METHODSPolyacrylamide-Agarose Gel Electrophoresis of RNA. Gels were prepared, essentially as described by Floyd et al. (13), in slabs 1.5 mm thick by 14 cm wide, and contained polyacrylamide (2.0-2.8%), 0.6% agarose, 6 M urea, 36 mM Tris-phosphate at pH 7.8, 1 mM EDTA. RNA samples for e...
A phosphotriester solid phase method on a polyamide support has been used to prepare oligodeoxyribonucleotides up to 12 units long. Compared to solid phase phosphodiester synthesis the new methodology is quicker, more flexible and gives 10-60-fold better overall yields.
A 514-base pair fragment of double-stranded DNA coding for human interferon-alpha 1 (166 amino acid residues), and containing initiation and termination signals plus appropriate restriction enzyme sites for plasmid insertion, has been totally synthesized. The synthesis involved preparation of 66 oligodeoxyribonucleotides, ranging in size from 14 to 21 residues, plus 1 deoxydecanucleotide, by rapid, solid phase procedures, and enzymatic ligation of the oligonucleotides. After ligation of the synthetic gene to a plasmid vector and transformation of Escherichia coli, clones containing the anticipated gene sequence were obtained.
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