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.

The reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of low-abundance mRNA, often obtained from limited tissue samples. However, it is a complex technique, there are substantial problems associated with its true sensitivity, reproducibility and specificity and, as a quantitative method, it suffers from the problems inherent in PCR. The recent introduction of fluorescence-based kinetic RT-PCR procedures significantly simplifies the process of producing reproducible quantification of mRNAs and promises to overcome these limitations. Nevertheless, their successful application depends on a clear understanding of the practical problems, and careful experimental design, application and validation remain essential for accurate quantitative measurements of transcription. This review discusses the technical aspects involved, contrasts conventional and kinetic RT-PCR methods for quantitating gene expression and compares the different kinetic RT-PCR systems. It illustrates the usefulness of these assays by demonstrating the significantly different levels of transcription between individuals of the housekeeping gene family, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).

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Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays.

In contrast to mouse epidermal cells, human skin keratinocytes are rather resistant to transformation in vitro. Immortalization has been achieved by SV40 but has resulted in cell lines with altered differentiation. We have established a spontaneously transformed human epithelial cell line from adult skin, which maintains full epidermal differentiation capacity. This HaCaT cell line is obviously immortal (greater than 140 passages), has a transformed phenotype in vitro (clonogenic on plastic and in agar) but remains nontumorigenic. Despite the altered and unlimited growth potential, HaCaT cells, similar to normal keratinocytes, reform an orderly structured and differentiated epidermal tissue when transplanted onto nude mice. Differentiation-specific keratins (Nos. 1 and 10) and other markers (involucrin and filaggrin) are expressed and regularly located. Thus, HaCaT is the first permanent epithelial cell line from adult human skin that exhibits normal differentiation and provides a promising tool for studying regulation of keratinization in human cells. On karyotyping this line is aneuploid (initially hypodiploid) with unique stable marker chromosomes indicating monoclonal origin. The identity of the HaCaT line with the tissue of origin was proven by DNA fingerprinting using hypervariable minisatellite probes. This is the first demonstration that the DNA fingerprint pattern is unaffected by long-term cultivation, transformation, and multiple chromosomal alterations, thereby offering a unique possibility for unequivocal identification of human cell lines. The characteristics of the HaCaT cell line clearly document that spontaneous transformation of human adult keratinocytes can occur in vitro and is associated with sequential chromosomal alterations, though not obligatorily linked to major defects in differentiation.

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Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.

The human genome contains many dispersed tandem-repetitive 'minisatellite' regions detected via a shared 10-15-base pair 'core' sequence similar to the generalized recombination signal (chi) of Escherichia coli. Many minisatellites are highly polymorphic due to allelic variation in repeat copy number in the minisatellite. A probe based on a tandem-repeat of the core sequence can detect many highly variable loci simultaneously and can provide an individual-specific DNA 'fingerprint' of general use in human genetic analysis.

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Hypervariable 'minisatellite' regions in human DNA.

Interspersed DNA elements of the form (dC-dA)n.(dG-dT)n constitute one of the most abundant human repetitive DNA families. We report that specific human (dC-dA)n.(dG-dT)n blocks are polymorphic in length among individuals and therefore represent a vast new pool of potential genetic markers. Comparison of sequences from the literature for (dC-dA)n.(dG-dT)n blocks cloned two or more times revealed length polymorphisms in seven of eight cases. Variations in the lengths of 10 (dC-dA)n.(dG-dT)n blocks were directly demonstrated by amplifying the DNA within and immediately flanking the repeat blocks by using the polymerase chain reaction and then resolving the amplified DNA on polyacrylamide DNA sequencing gels. Use of the polymerase chain reaction to detect DNA polymorphisms offers improved sensitivity and speed compared with standard blotting and hybridization.

Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction

Simple tandem-repetitive regions of DNA (or 'minisatellites') which are dispersed in the human genome frequently show substantial length polymorphism arising from unequal exchanges which alter the number of short tandem repeats in a minisatellite. We have shown previously that the repeat elements in a subset of human minisatellites share a common 10-15-base-pair (bp) 'core' sequence which might act as a recombination signal in the generation of these hypervariable regions. A hybridization probe consisting of the core repeated in tandem can detect many highly polymorphic minisatellites simultaneously to provide a set of genetic markers of general use in human linkage analysis. We now show that other variant (core)n probes can detect additional sets of hypervariable minisatellites to produce somatically stable DNA 'fingerprints' which are completely specific to an individual (or to his or her identical twin) and can be applied directly to problems of human identification, including parenthood testing.

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Individual-specific 'fingerprints' of human DNA.

Tandem-repetitive minisatellite regions in vertebrate DNA frequently show substantial allelic variation in the number of repeat units. This variation is thought to arise through processes such as unequal crossover or replication slippage1–12. We show here that the spontaneous mutation rate to new length alleles at extremely variable human minisatellites is sufficiently high to be directly measurable in human pedigrees. The mutation rate at different loci increases with variability in accord with the neutral mutation/random drift hypothesis, and rises to 5% per gamete for the most unstable human minisatellite isolated. Mutations are sporadic, occur with similar frequencies in sperm and oocytes, and can involve the gain or loss of substantial numbers of repeat units, consistent with length changes arising primarily by unequal exchange at meiosis. Germline instability must therefore be taken into account when using hypervariable loci as genetic markers, particularly in pedigree analysis and parenthood testing.

Spontaneous mutation rates to new length alleles at tandem-repetitive hypervariable loci in human DNA.

Hypervariable minisatellites can be amplified from human DNA by the polymerase chain reaction, using primers from DNA flanking the minisatellite to amplify the entire block of tandem repeat units. Minisatellite alleles up to 5-10 kb long can be faithfully amplified. At least six minisatellite loci can be co-amplified from the same DNA sample and simultaneously detected to provide a reproducible and highly variable DNA fingerprint which can be obtained from nanogram quantities of human DNA. The polymerase chain reaction can also be used to analyse single target minisatellite molecules and single human cells, despite the appearance of spurious PCR products from some hypervariable loci. DNA fingerprinting at the level of one or a few cells therefore appears possible.

Amplification of human minisatellites by the polymerase chain reaction: towards DNA fingerprinting of single cells

https://www.cell.com/cell/pdf/0092-8674(90)90598-9.pdf

Victoria Wilson

Papers

Hypervariable 'minisatellite' regions in human DNA.

Individual-specific 'fingerprints' of human DNA.

Spontaneous mutation rates to new length alleles at tandem-repetitive hypervariable loci in human DNA.

Amplification of human minisatellites by the polymerase chain reaction: towards DNA fingerprinting of single cells

Repeat unit sequence variation in minisatellites: a novel source of DNA polymorphism for studying variation and mutation by single molecule analysis.