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

Two new methods were used to establish a rapid and highly sensitive prenatal diagnostic test for sickle cell anemia. The first involves the primer-mediated enzymatic amplification of specific beta-globin target sequences in genomic DNA, resulting in the exponential increase (220,000 times) of target DNA copies. In the second technique, the presence of the beta A and beta S alleles is determined by restriction endonuclease digestion of an end-labeled oligonucleotide probe hybridized in solution to the amplified beta-globin sequences. The beta-globin genotype can be determined in less than 1 day on samples containing significantly less than 1 microgram of genomic DNA.

https://science.sciencemag.org/content/sci/230/4732/1350.full.pdf

Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia.

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

Specific alleles at the HLA-DR and -DQ loci have been associated with a variety of autoimmune diseases. The role of HLA-DP polymorphism in susceptibility has not been as fully explored as that of the other class II antigens due to the complexity of the primed lymphocyte typing (PLT) method for determining DPw specificities. These class II loci encode a heterodimer consisting of an alpha (ca 32 kDa) and a beta (ca 29 kDa) glycoprotein chain. These α-β heterodimers are highly polymorphic transmembrane proteins which bind peptides derived from the processing of foreign antigens (Babbitt et al., 1985; Guillet et al., 1987; Sette et al., 1987; Unanue et al., 1987; Watts et al., 1986). The activation of CD4+ T lymphocytes results from the recognition of this class II-peptide complex by the alpha-beta T cell receptor. In addition to their role in peptide binding on antigen-presenting cells, the polymorphic class II molecules present on thymic stromal cells influence the specificity of the mature T cell repertoire (Kappler et al., 1987; Kappler et al., 1988; MacDonald et al., 1988; Teh et al., 1988).

HLA Class II Sequence Polymorphism and Autoimmunity

Class II mutant B lymphoblastoid cell lines (B-LCL) affected in presentation of soluble antigen to HLArestricted antigen-specific T cells can be used to characterize the molecular interactions involved in antigen presentation. We have isolated class II mutants in the DR3 haplotype, a haplotype of particular interest because of its association with several autoimmune diseases (1). Here, we describe the use of antigen loss mutants to localize restriction elements to the DRβ1, DRβ3, and non-DR dimers of the DR3 haplotype, and the use of mutants expressing altered DR3 molecules to identify regions of DR3 molecules involved in antigen presentation and in α/β chain association.

Henry A. Erlich

Papers

HLA Class II Sequence Polymorphism and Autoimmunity

Functional Analysis of HLA-DR3: A Mutational Approach