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.

The evolution of allelic diversity at the primate major histocompatibility complex class II loci.

Shared molecular markers of genetic predisposition to seropositive rheumatoid arthritis.

HLA and insulin-dependent diabetes mellitus.

A variety of different techniques have been used to characterize the polymorphism within the HLA class II molecules; traditionally these included serology, cellular assays such as the mixed lymphocyte reaction, and twodimensional gel electrophoresis. Recently, DNA-based methodologies including restriction fragment length polymorphism analysis as well as cloning and sequence analysis have gained widespread use (for a review see Silver 1990). The latter has been greatly facilitated by the development of the polymerase chain reaction (PCR; Saiki et al. 1985, 1988; Mullis and Faloona 1987). Using these techniques, 34 different DRB1 alleles have been reported to date (Marsh and Bodmer 1990). We now report the sequence of a new DRB1 allele, DRBI*LYIO, and its haplotypic association. The initial discovery of the allele was made during an HLA analysis of a patient with lyme arthritis (G. Ruberti, A.B. Begovich, A.C. Steere, W. Klitz, H .A. Erlich, C.G. Fathman, manuscript submitted for publication). Genomic DNA was enzymatically amplified using the PCR with primers specific for the DRB1 locus (S. Scharf, R.L. Griffith, H .A. Erlich, manuscript submitted for publication). Analysis of the PCR product with nonradioactive DRB sequence-specific oligonucleotide (SSO) probes in a dot-blot format (Erlich et al. 1986; Levenson and Chang 1990; Scharf et al. manuscript submitted for publication) revealed a new hybridization pattern which suggested that the individual carried the DRB1*0402 (DR4DwlO) allele on one chromosome and a new allele on the other chromosome. The oligonucleotide hybridization pattern suggested that the new allele was a hybrid of two previously described DRB1 alleles; it had regions of sequences similar to DRB1 \"0801, *0802, *0803, and \"1201 at the 5' end and to \"1401 at

DRB1*LY10--a new DRB1 allele and its haplotypic association.

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.

Henry A. Erlich

Papers

The evolution of allelic diversity at the primate major histocompatibility complex class II loci.

Shared molecular markers of genetic predisposition to seropositive rheumatoid arthritis.

HLA and insulin-dependent diabetes mellitus.

DRB1*LY10--a new DRB1 allele and its haplotypic association.

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