Fast and sensitive silver staining of DNA in polyacrylamide gels

Edited by the inventor of polymerase chain reaction (PCR) and the 1993 Nobel Prize winner in Chemistry, Kary Mullis, as well as two experts in the field, this handbook provides up-to-date methodological protocols from the world's leading laboratories, in addition to new techniques and enhanced applications not yet available in book form. Nearly 40 chapters inform the novice and experienced PCR user on how to optimize their results. In the chapters on applications, researchers provide not only protocols, but also descriptions of how PCR has revolutionized their particular field. Future enhancements of PCR, as well as new potential uses, are discussed. Readers can learn how PCR has changed the face of diagnostic testing, cancer research, genetics, forensics, plant biology, DNA sequencing and gene therapy. Special sections include the latest on QPCR, non-isotopic detection, genetic analysis, and PCR and the world of business, which includes a behind-the-scenes look at the legal battles between biotech giants Cetus and DuPont. It also provides insights into the origins of PCR and the history of nucleic acid research.

The polymerase chain reaction

We have developed a new method for forensic identification of individuals, in which a panel of biallelic DNA markers are amplified by the PCR, and the variable nucleotides are detected in the amplified DNA fragments by the solid-phase minisequencing method. A panel of 12 common polymorphic nucleotides located on different chromosomes with reported allele frequencies close to .5 were chosen for the test. The allele frequencies for most of the markers were found to be similar in the Finnish and other Caucasian populations. We also introduce a novel approach for rapid determination of the population frequencies of biallelic markers. By this approach we were able to determine the allele frequencies of the markers in the Finnish population, by quantitative analysis of three pooled DNA samples representing 3,000 individuals. The power of discrimination and exclusion of the solid-phase minisequencing typing test with 12 markers was similar to that of three VNTR markers that are routinely used in forensic analyses at our institute. The solid-phase minisequencing method was successfully applied to type paternity and forensic case samples. We also show that the quantitative nature of our method allows typing of mixed samples.

Identification of individuals by analysis of biallelic DNA markers, using PCR and solid-phase minisequencing.

Molecular Genetics of Hypervariable DNA.- Principles and recent advances in human DNA fingerprinting.- Generation of variability at VNTR loci in human DNA.- Human VNTR sequences in porcine HTF-islands.- Oligonucleotide fingerprinting using simple repeat motifs: a convenient, ubiquitously applicable method to detect hypervariability for multiple purposes.- DNA fingerprinting of the human intestinal parasite Giardia intestinalis with hypervariable minisatellite sequences.- Human variable number of tandem repeat probes as a source of polymorphic markers in experimental animals.- DNA Fingerprinting: the utilization of minisatellite probes to detect a somatic mutation in the Proteus syndrome.- Genetic variability of satellite sequence in the dipteran Musca domestica.- Population Genetics and Evolutionary Biology.- Analysis of population genetic structure by DNA fingerprinting.- Population genetics of hypervariable loci: analysis of PCR based VNTR polymorphism within a population.- Population genetic data determined for five different single locus minisatellite probes.- Multilocus and single locus minisatellite analysis in population biological studies.- Helpers-at-the-nest in European Bee-eaters (Merops apiaster): a genetic analysis.- Cloning, characterization and evolution of Indian peafowl Pavo christatus minisatellite loci.- Use of sex-linked minisatellite fragments to investigate genetic differentiation and migration of North American populations of the peregrine falcon (Falco peregrinus).- Economically-important Animals and Plants.- Hypervariable DNA markers and their applications in the chicken.- Cloning of hypervariable minisatellite and simple sequence microsatellite repeats for DNA fingerprinting of important aquacultural species of salmonids and tilapias.- Genetic factors accountable for line-specific DNA fingerprint bands in quail.- Identification of markers associated with quantitative trait loci in chickens by DNA fingerprinting.- Two dimensional DNA-fingerprinting in animals.- Applications of DNA fingerprinting in plant breeding.- Oligonucleotide fingerprinting in plants and fungi.- The isolation and characterisation of plant sequences homologous to human hypervariable minisatellites.- Implementation of DNA Typing.- DNA fingerprinting: a biotechnology in business.- DNA fingerprinting: its applications in forensic case work.- Tracking the violent criminal offender through DNA typing profiles - a national database system concept.- The quality control of cell banks using DNA fingerprinting.- Detection of amplified VNTR alleles by direct chemiluminescence: application to the genetic identification of biological samples in forensic cases.- Genetic typing using automated electrophoresis and fluorescence detection.

DNA Fingerprinting: Approaches and Applications

A series of experiments has been performed to evaluate amplification and typing of the D1S80 VNTR locus. The validation study that has been carried out showed that correct D1S80 typing results can be obtained when a defined amplification protocol and a high-resolution polyacrylamide gel electrophoresis method are used. The use of the Chelex extraction protocol has substantially reduced the processing time. DNA-extraction, amplification and subsequent typing can be performed in one day. The discrimination power of this locus is 0.94 in a Dutch Caucasian population sample. The system is extremely sensitive: 0.1 ng of genomic DNA gave a correct typing result. The test could also detect the correct genotypes in mixed samples containing DNA from different individuals. Even if the major type was in a 20-fold excess, the minority type could still be amplified and typed correctly. We have found no deviation from Hardy-Weinberg equilibrium in a Dutch Caucasian population sample. Evidence for the somatic stability of this locus was obtained from a set of experiments where we compared DNA-profiles from corresponding blood, semen and saliva samples. The results of this study suggest that in the near future analysis of the D1S80 locus by DNA-amplification can be applied in actual forensic case work.

PCR-amplification and detection of the human D1S80 VNTR locus Amplification conditions, population genetics and application in forensic analysis

Separation of polymerase chain reaction (PCR) amplification of specific fragment length polymorphisms was carried out on rehydratable polyacrylamide gels on a horizontal flat slab system. A discontinuous sulfate-borate buffer system was employed on 5-8% T gels crosslinked with 3.5% C. Samples were diluted in leading sulfate ion buffer at 1/10 the ionic strength of the separating gel buffer and placed directly onto the surface of the rehydrated gels in 0.5-10 microliters volumes. The trailing ion and counterion were contained in a gel plug and placed directly onto the anodal and cathodal ends of the gel, and the electrodes placed directly onto the surface of the gel plugs. Filter paper wicks, soaked in diluted leading ion buffer, were placed along each side to lower the ionic strength of the edges, thereby increasing mobility at the edge and thus preventing smile effects. The gel-gel contact of the plug and separating gel prevent the production of a junction potential which occurs between dissimilar materials such as a paper wick and the gel. Ten- to 20-cm separations were carried out from 2-5 h, respectively, and resolution in the 20 cm system was 1.6-4 bp (base pairs) between 100 and 500 bp, 4-7 bp between 500 and 1000 bp, 12-20 bp between 1000 and 2000 bp and about 50 bp between 2000 and 3000 bp. Between 3000 and 4000 bp, resolution fell off to +/- 100 bp. Sensitivity, using a silver stain, indicated that one could readily distinguish less than 10 pg of DNA per mm width on the gels.(ABSTRACT TRUNCATED AT 250 WORDS)

Polymerase chain reaction amplification products separated on rehydratable polyacrylamide gels and stained with silver

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Polymerase chain reaction amplification products separated on rehydratable polyacrylamide gels and stained with silver