A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme, isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified. Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule present only once in a sample of 10(5) cells.

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Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase

A novel DNA fingerprinting technique called AFLP is described. The AFLP technique is based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA. The technique involves three steps: (i) restriction of the DNA and ligation of oligonucleotide adapters, (ii) selective amplification of sets of restriction fragments, and (iii) gel analysis of the amplified fragments. PCR amplification of restriction fragments is achieved by using the adapter and restriction site sequence as target sites for primer annealing. The selective amplification is achieved by the use of primers that extend into the restriction fragments, amplifying only those fragments in which the primer extensions match the nucleotides flanking the restriction sites. Using this method, sets of restriction fragments may be visualized by PCR without knowledge of nucleotide sequence. The method allows the specific co-amplification of high numbers of restriction fragments. The number of fragments that can be analyzed simultaneously, however, is dependent on the resolution of the detection system. Typically 50-100 restriction fragments are amplified and detected on denaturing polyacrylamide gels. The AFLP technique provides a novel and very powerful DNA fingerprinting technique for DNAs of any origin or complexity.

AFLP: a new technique for DNA fingerprinting.

We describe a new molecular approach to analyzing the genetic diversity of complex microbial populations. This technique is based on the separation of polymerase chain reaction-amplified fragments of genes coding for 16S rRNA, all the same length, by denaturing gradient gel electrophoresis (DGGE). DGGE analysis of different microbial communities demonstrated the presence of up to 10 distinguishable bands in the separation pattern, which were most likely derived from as many different species constituting these populations, and thereby generated a DGGE profile of the populations. We showed that it is possible to identify constituents which represent only 1% of the total population. With an oligonucleotide probe specific for the V3 region of 16S rRNA of sulfate-reducing bacteria, particular DNA fragments from some of the microbial populations could be identified by hybridization analysis. Analysis of the genomic DNA from a bacterial biofilm grown under aerobic conditions suggests that sulfate-reducing bacteria, despite their anaerobicity, were present in this environment. The results we obtained demonstrate that this technique will contribute to our understanding of the genetic diversity of uncharacterized microbial populations.

Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA

Phylogenetic identification and in situ detection of individual microbial cells without cultivation.

Subpopulations of RNA molecules that bind specifically to a variety of organic dyes have been isolated from a population of random sequence RNA molecules. Roughly one in 10(10) random sequence RNA molecules folds in such a way as to create a specific binding site for small ligands.

In vitro selection of RNA molecules that bind specific ligands.

The invention is embodied by digesting a vector DNA sequence with a restriction enzyme, removing any unwanted nucleotides, treating the cleaved vector with exonuclease III to create 5′ sticky ends, mixing with the thus treated vector an even number of single-­stranded nucleotides of alternating sense and anti-­sense sequence which alternately encode or are complementary to the desired sequences in the product DNA, and which single-stranded ologinucleotides have termini which overlap and are complementary to termini of the adjacent oligonucleotide(s) and/or cleaved vector 5′ sticky ends so as to form a tendem bridge between the 5′ sticky end upstream of the original cleavage site and the 5′ sticky end downstream of the original cleavage site, and treating the mixture with DNA polyemrase and ligase.

Modification of DNA sequences

DRβ sequence polymorphism has been analyzed using the polymerase chain reaction in vitro amplification method. The amplified DRβ segments have been cloned and sequenced to determine the extent of allotypic (allele) and isotypic (locus) variation. The sequences have been assigned to either the DRβ 1 or the second DRβ chain locus on specific haplotypes. This sequence information can serve as the basis for a rapid DNA typing method using a dot-blot format with sequence-specific oligonucleotide probes. The patchwork pattern of polymorphism revealed by this analysis may reflect gene conversion-like mechanisms between different alleles and different loci. A previously unreported sequence, termed DRβX, is described and helps define one of the three major evolutionary groups of DR haplotypes.

Analysis of Isotypic and Allotypic Sequence Variation in the HLA-DRβ Region Using the In Vitro Enzymatic Amplification of Specific DNA Segments

The invention provides an oligonucleotide primer exhibiting complementarity to an end of a nucleic acid sequence corresponding to a second exon sequence in an HLA Class II gene, wherein said primer can be hybridized to said sequence and then extended thereon to produce a single stranded extension product which includes a sequence complementary to said second exon sequence. Such primers can be used in a nucleic acid amplification process to enable detection of gene variations or polymorphism. For detection purposes, the invention also includes an oligonucleotide probe that is capable of hybridizing to a sequence in the second exon of an HLA Class II gene so as to permit detection of said sequence when said probe is labelled as so-hybridized.

Primers and probes for nucleic acid detection

Recombinant methods for the production of Ricin A, Ricin B, Ricin of Diphtheria toxin (DT)A or AB′ fragment are described together with suitable hosts and vectors. Immunotoxin conjugates comprising Ricin toxin A chain or Diphtheria toxin are also described.

Recombinant methods for the production of ricin a, ricin b, ricin or diphtheria toxin (dt)a or ab' fragment, suitable hosts and vectors therefor, and conjugates comprising ricin toxin a chain or diphtheria toxin

Recombinant vectors and methods for constructing ricin B are disclosed. The coding sequence for ricin B was cloned, disposed in suitable expression vectors and produced free of components normally accompanying this peptide. In addition, a novel means of reconstructing missing portions of coding sequence, and certain improvements in messenger RNA purification are disclosed.

Glenn Thomas Horn

Papers

Modification of DNA sequences

Analysis of Isotypic and Allotypic Sequence Variation in the HLA-DRβ Region Using the In Vitro Enzymatic Amplification of Specific DNA Segments

Primers and probes for nucleic acid detection

Recombinant methods for the production of ricin a, ricin b, ricin or diphtheria toxin (dt)a or ab' fragment, suitable hosts and vectors therefor, and conjugates comprising ricin toxin a chain or diphtheria toxin

Recombinant ricin fragments, vectors and transformed hosts expressing the same, the modification of DNA sequences, and isolation of mRNA