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.

PROBLEM TO BE SOLVED: To obtain a kit for detecting the subject sequence in a sample by combining a pair of primers exponentially amplifying each nucleic acid containing a different change to be detected with a probe specific to a nucleic acid to be detected and a membrane for immobilizing an amplified product. SOLUTION: This kit for detecting a specific nucleotide sequence comprises two kinds of oligomer nucleotide primers for each nucleic acid containing a different change to be detected, in which an extended product synthesized from one primer, in the case of the extended product being separated from its complementary body, can function as a template for synthesizing an extended product of another primer and the primers can exponentially amplify the nucleic acid, a sequence specific probe which can be hybridized with the amplified nucleic acid sequence only in the case of a probe sequence being complementary to a certain region of the amplified sequence and has about 15 to about 25 base length striding over a nucleotide change to be detected and specific to the nucleotide change to be detected and a membrane to be connected to the amplified nucleic acid.

Method and kit for detecting specific nucleotide sequence

Gereinigtes thermostabiles enzym und verfahren zur verstaerkung, zum nachweis und/oder zur klonierung von nukleinsaeuresequenzen durch verwendung dieses enzyms.

Detection of specific nucleotide variations and genetic polymorphisms in nucleic acids

The autoimmune dermatologic disease pem- phigus vulgaris (PV) is associated with the HLA serotypes DR4 and DRw6. Susceptibility to PV could be conferred either by sequences shared between the DR4 and DRw6 haplotypes or by different sequences in these haplotypes. We have examined the distribution of DR and DQ fl-chain and DQ a-chain alleles in PV patients and In control subjects by hybridization with oligonucleotide probes and sequence analysis of in vitro ampli- fied DNA. Ninety percent (34/38) of the DR4 haplotypes in patients contain a specific DRflI sequence present in 36% (16 of 44) of DR4 controls (P = 0.001). This sequence is also found in DRw6 haplotypes. However, it is present in only 25% (6 of 24) of DRw6 patients. The results of our analysis indicate that predisposition to PV is conferred by different sequences In DR4 and DRw6 haplotypes. The DR4 susceptibility is highly asso- ciated with the DwlO DRIlI allele, implicating the polymorphic residues in the third hypervariable region. The DRw6 suscep-

HLA class II allelic variation and susceptibility to pemphigus vulgaris (major histocompatibility complex/autolmmunlty/polymerase chain reaction/sequence polymorphism/oligonucleotide probes)

The pattern of nucleotide sequence variation in the HLA-DQ genes has been analyzed with respect to the known serologically defined HLA types and to susceptibility to insulin-dependent diabetes (IDDM). Thus far, 8 allelic variants have been found for the DQα gene, and 12 alleles have been seen for DQβ. The DQw1 specificity correlates best with a related set of DQα variants, whereas the DQw2, DQw3, and DQ(blank) specificities correlate with alleles of DQβ. A pattern of amino acid substitutions at position 57 in the DQβ protein appears to reflect the degree of susceptibility to IDDM for the different HLA haplotypes. This portion of DQβ also shows significant homology with several human viruses, suggesting that it may be a preferred site for molecular mimicry.

Allelic Sequence Variation in the HLA-DQ Region: Implications for Localization of Serological Specificities and for Susceptibility to Autoimmune Disease

Glenn Thomas Horn

Papers

Method and kit for detecting specific nucleotide sequence

Gereinigtes thermostabiles enzym und verfahren zur verstaerkung, zum nachweis und/oder zur klonierung von nukleinsaeuresequenzen durch verwendung dieses enzyms.

Detection of specific nucleotide variations and genetic polymorphisms in nucleic acids

HLA class II allelic variation and susceptibility to pemphigus vulgaris (major histocompatibility complex/autolmmunlty/polymerase chain reaction/sequence polymorphism/oligonucleotide probes)

Allelic Sequence Variation in the HLA-DQ Region: Implications for Localization of Serological Specificities and for Susceptibility to Autoimmune Disease