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.

Fast and sensitive silver staining of DNA in polyacrylamide gels

The utility of RFLP (restriction fragment length polymorphism), RAPD (random-amplified polymorphic DNA), AFLP (amplified fragment length polymorphism) and SSR (simple sequence repeat, microsatellite) markers in soybean germplasm analysis was determined by evaluating information content (expected heterozygosity), number of loci simultaneously analyzed per experiment (multiplex ratio) and effectiveness in assessing relationships between accessions. SSR markers have the highest expected heterozygosity (0.60), while AFLP markers have the highest effective multiplex ratio (19). A single parameter, defined as the marker index, which is the product of expected heterozygosity and multiplex ratio, may be used to evaluate overall utility of a marker system. A comparison of genetic similarity matrices revealed that, if the comparison involved both cultivated (Glycine max) and wild soybean (Glycine soja) accessions, estimates based on RFLPs, AFLPs and SSRs are highly correlated, indicating congruence between these assays. However, correlations of RAPD marker data with those obtained using other marker systems were lower. This is because RAPDs produce higher estimates of interspecific similarities. If the comparisons involvedG. max only, then overall correlations between marker systems are significantly lower. WithinG. max, RAPD and AFLP similarity estimates are more closely correlated than those involving other marker systems.

The comparison of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis

Recent advances in the application of the polymerase chain reaction make it possible to score individuals at a large number of loci The RAPD (random amplified polymorphic DNA) method is one such technique that has attracted widespread interest The analysis of population structure with RAPD data is hampered by the lack of complete genotypic information resulting from dominance, since this enhances the sampling variance associated with single loci as well as induces bias in parameter estimation We present estimators for several population-genetic parameters (gene and genotype frequencies, within- and between-population heterozygosities, degree of inbreeding and population subdivision, and degree of individual relatedness) along with expressions for their sampling variances Although completely unbiased estimators do not appear to be possible with RAPDs, several steps are suggested that will insure that the bias in parameter estimates is negligible To achieve the same degree of statistical power, on the order of 2 to 10 times more individuals need to be sampled per locus when dominant markers are relied upon, as compared to codominant (RFLP, isozyme) markers Moreover, to avoid bias in parameter estimation, the marker alleles for most of these loci should be in relatively low frequency Due to the need for pruning loci with low-frequency null alleles, more loci also need to be sampled with RAPDs than with more conventional markers, and some problems of bias cannot be completely eliminated

Analysis of population genetic structure with RAPD markers

Polyphasic taxonomy, a consensus approach to bacterial systematics.

The surprising finding that amplification of genomic DNA can be directed by only one oligonucleotide primer of arbitrary sequence to produce a characteristic spectrum of short DNA products of varying complexity, was applied as a strategy to detect genetic differences between organisms. This approach, DNA amplification fingerprinting (DAF), does not depend on cloning or DNA sequence information and can generate fingerprints from DNA of viral, bacterial, fungal, plant and animal origins. Primers as short as 5 nucleotides in length can produce complex banding patterns that are resolved by polyacrylamide gel electrophoresis and silver staining. Amplification fragment length polymorphisms (AFLPs) were detected between different human individuals as well as between soybean cultivars. It is anticipated that DAF will have wide application for DNA analysis.

DNA amplification fingerprinting using very short arbitrary oligonucleotide primers.

This protocol describes a simple silver staining method used to visualize DNA fragments and other organic molecules with unsurpassed detail following traditional polyacrylamide gel electrophoresis (PAGE). Sensitivity rivals radioisotopic methods and DNA in the picogram range can be reliably detected. The described protocol is fast (approximately 1 h) and is implemented using readily available chemicals and materials. To achieve the sensitivity and visual clarity expected, quality reagents and clean handling are important. The updated protocol described here is based on the widely used method of Bassam et al. (1991), but provides improved image contrast and less risk of staining artefacts.

Silver staining DNA in polyacrylamide gels

Nucleic acids can be detected at the picogram level using a quick and simple silver staining method (2). Using very thin polyesterbacked polyacrylamide gels, a further simplified protocol was compared to other widely used silver staining procedures. The improved protocol described here was the most sensitive, the fastest to perform, and had relatively few steps and reagents. This method also produced the least number of staining artifacts and offered images of high contrast.

Silver staining of DNA in polyacrylamide gels

DNA amplification fingerprinting (DAF) is the enzymatic amplification of arbitrary stretches of DNA which is directed by very short oligonucleotide primers of arbitrary sequence to generate complex but characteristic DNA fingerprints. To determine the contribution of primer sequence and length to the fingerprint pattern and the effect of primer-template mismatches, DNA was amplified from several sources using sequence-related primers. Primers of varying length, constructed by removing nucleotides from the 5′ terminus, produced unique patterns only when primers were 8 nucleotides or fewer in length. Larger primers produced either identical or related fingerprints, depending on the sequence. Single base changes within this first 8-nucleotide region of the primer significantly altered the spectrum of amplification products, especially at the 3′ terminus. Increasing annealing temperatures from 15° to 70° C during amplification did not shift the boundary of the 8-nucleotide region, but reduced the amplification ability of shorter primers. Our observations define a 3′-terminal oligonucleotide domain that is at least 8 bases in length and largely conditions amplification, but that is modulated by sequences beyond it. Our results indicate that only a fraction of template annealing sites are efficiently amplified during DAF. A model is proposed in which a single primer preferentially amplifies certain products due to competition for annealing sites between primer and terminal hairpin loop structures of the template.

Brant J. Bassam

Papers

Fast and sensitive silver staining of DNA in polyacrylamide gels

DNA amplification fingerprinting using very short arbitrary oligonucleotide primers.

Silver staining DNA in polyacrylamide gels

Silver staining of DNA in polyacrylamide gels

Primer-template interactions during DNA amplification fingerprinting with single arbitrary oligonucleotides.