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.

Simple and reproducible fingerprints of complex genomes can be generated using single arbitrarily chosen primers and the polymerase chain reaction (PCR). No prior sequence information is required. The method, arbitrarily primed PCR (AP-PCR), involves two cycles of low stringency amplification followed by PCR at higher stringency. We show that strains can be distinguished by comparing polymorphisms in genomic fingerprints. The generality of the method is demonstrated by application to twenty four strains from five species of Staphylococcus, eleven strains of Streptococcus pyogenes and three varieties of Oryza sativa (rice).

Fingerprinting genomes using PCR with arbitrary primers

Effective methods are needed to identify and isolate those genes that are differentially expressed in various cells or under altered conditions. This report describes a method to separate and clone individual messenger RNAs (mRNAs) by means of the polymerase chain reaction. The key element is to use a set of oligonucleotide primers, one being anchored to the polyadenylate tail of a subset of mRNAs, the other being short and arbitrary in sequence so that it anneals at different positions relative to the first primer. The mRNA subpopulations defined by these primer pairs were amplified after reverse transcription and resolved on a DNA sequencing gel. When multiple primer sets were used, reproducible patterns of amplified complementary DNA fragments were obtained that showed strong dependence on sequence specificity of either primer.

https://science.sciencemag.org/content/sci/257/5072/967.full.pdf

Differential Display of Eukaryotic Messenger RNA by Means of the Polymerase Chain Reaction

We developed bulked segregant analysis as a method for rapidly identifying markers linked to any specific gene or genomic region. Two bulked DNA samples are generated from a segregating population from a single cross. Each pool, or bulk, contains individuals that are identical for a particular trait or genomic region but arbitrary at all unlinked regions. The two bulks are therefore genetically dissimilar in the selected region but seemingly heterozygous at all other regions. The two bulks can be made for any genomic region and from any segregating population. The bulks are screened for differences using restriction fragment length polymorphism probes or random amplified polymorphic DNA primers. We have used bulked segregant analysis to identify three random amplified polymorphic DNA markers in lettuce linked to a gene for resistance to downy mildew. We showed that markers can be reliably identified in a 25-centimorgan window on either side of the targeted locus. Bulked segregant analysis has several advantages over the use of near-isogenic lines to identify markers in specific regions of the genome. Genetic walking will be possible by multiple rounds of bulked segregation analysis; each new pair of bulks will differ at a locus identified in the previous round of analysis. This approach will have widespread application both in those species where selfing is possible and in those that are obligatorily outbreeding.

https://www.pnas.org/content/pnas/88/21/9828.full.pdf

Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations.

We constructed nine sets of oligonucleotide primers on the basis of the results of DNA hybridization of cloned genes from Neurospora crassa and Aspergillus nidulans to the genomes of select filamentous ascomycetes and deuteromycetes (with filamentous ascomycete affiliations). Nine sets of primers were designed to amplify segments of DNA that span one or more introns in conserved genes. PCR DNA amplification with the nine primer sets with genomic DNA from ascomycetes, deuteromycetes, basidiomycetes, and plants revealed that five of the primer sets amplified a product only from DNA of the filamentous ascomycetes and deuteromycetes. The five primer sets were constructed from the N. crassa genes for histone 3, histone 4, beta-tubulin, and the plasma membrane ATPase. With these five primer sets, polymorphisms were observed in both the size of and restriction enzyme sites in the amplified products from the filamentous ascomycetes. The primer sets described here may provide useful tools for phylogenetic studies and genome analyses in filamentous ascomycetes and deuteromycetes (with ascomycete affiliations), as well as for the rapid differentiation of fungal species by PCR.

Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes.

Molecular genetic maps are commonly constructed by analyzing the segregation of restriction fragment length polymorphisms (RFLPs) among the progeny of a sexual cross. Here we describe a new DNA polymorphism assay based on the amplification of random DNA segments with single primers of arbitrary nucleotide sequence. These polymorphisms, simply detected as DNA segments which amplify from one parent but not the other, are inherited in a Mendelian fashion and can be used to construct genetic maps in a variety of species. We suggest that these polymorphisms be called RAPD markers, after Random Amplified Polymorphic DNA.

/pdf/dna-polymorphisms-amplified-by-arbitrary-primers-are-useful-3dtvzw1rfi.pdf

DNA polymorphisms amplified by arbitrary primers are useful as genetic markers

A population of Arabidopsis thaliana recombinant inbred lines was constructed and used to develop a high-density genetic linkage map containing 252 random amplified polymorphic DNA markers and 60 previously mapped restriction fragment length polymorphisms. Linkage groups were correlated to the classical genetic map by inclusion of nine phenotypic markers in the mapping cross. We also applied a technique for local mapping that allows targeting of markers to a selected genome region by pooling DNA from recombinant inbred lines based on their genotype. We conclude that random amplified polymorphic DNAs, used in conjunction with a recombinant inbred population, can facilitate the genetic and physical characterization of the Arabidopsis genome and that this method is generally applicable to other organisms for which appropriate populations either are available or can be developed.

https://www.pnas.org/content/pnas/89/4/1477.full.pdf

Global and local genome mapping in Arabidopsis thaliana by using recombinant inbred lines and random amplified polymorphic DNAs

The genes encoding the two subunits (alpha and beta) of the cytochrome b559 (cyt b559) protein, psbE and psbF, were cloned from the unicellular, transformable cyanobacterium, Synechocystis 6803. Cyt b559, an intrinsic membrane protein, is a component of photosystem II, a membrane-protein complex that catalyzes photosynthetic oxygen evolution. However, the role of cyt b559 in photosynthetic electron transport is yet to be determined. A high degree of homology was found between the cyanobacterial and green plant chloroplastidic psbE and psbE genes and in the amino acid sequences of their corresponding protein products. Cartridge mutagenesis techniques were used to generate a deletion mutant of Synechocystis 6803 in which the psbE and psbF genes were replaced by a kanamycin-resistance gene cartridge. Physiological analyses indicated that the PSII complexes of the mutant were inactivated. We conclude that cyt b559 is an essential component of PSII.

https://www.embopress.org/doi/pdf/10.1002/j.1460-2075.1988.tb02816.x

Targeted mutagenesis of the psbE and psbF genes blocks photosynthetic electron transport: evidence for a functional role of cytochrome b559 in photosystem II.

The Photosystem II protein CP-47 has been hypothesized to be involved in binding the reaction center chlorophyll. The psbB gene, encoding this protein, was cloned from the genome of the cyanobacterium Synechocystis 6803, and sequenced. The DNA sequence is 68% homologous with that of the psbB gene from spinach, whereas the predicted amino acid sequence is 76% homologous. The hydropathy patterns of Synechocystis and spinach CP-47 are almost indistinguishable, indicating the same general CP-47 folding pattern in the thylakoid membrane in the two species. There are five pairs of histidine residues in CP-47 that are spaced by 13 or 14 amino acids and that are located in hydrophobic regions of the protein; these histidine residues may be involved in chlorophyll binding. Interruption of the psbB gene by a DNA fragment carrying a gene conferring kanamycin resistance results in a loss of Photosystem II activity. This indicates that an intact CP-47 is required for a functional Photosystem II complex, but does not necessarily indicate that this protein would house the reaction center.

Sequencing and modification of psbB, the gene encoding the CP-47 protein of Photosystem II, in the cyanobacterium Synechocystis 6803.

A process is provided for detecting polymorphisms on the basis of nucleotide differences in random segments of the nucleic acid by performing a primer extension reaction on the nucleic acids and comparing the extension reaction products. The random nucleic acid segment can be amplified by first dissociating the extension product from the template and contacting the dissociated extension product with a primer under conditions such that an amplification extension product is synthesized using the dissociated extension product as a template. Differences in the extension product are useful as markers in constructing genetic maps and as markers to distinguish or identify individuals.

John G. K. Williams

Papers

DNA polymorphisms amplified by arbitrary primers are useful as genetic markers

Global and local genome mapping in Arabidopsis thaliana by using recombinant inbred lines and random amplified polymorphic DNAs

Targeted mutagenesis of the psbE and psbF genes blocks photosynthetic electron transport: evidence for a functional role of cytochrome b559 in photosystem II.

Sequencing and modification of psbB, the gene encoding the CP-47 protein of Photosystem II, in the cyanobacterium Synechocystis 6803.

Process for detecting polymorphisms on the basis of nucleotide differences