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.

Procedures utilizing Chelex 100 chelating resin have been developed for extracting DNA from forensic-type samples for use with the PCR. The procedures are simple, rapid, involve no organic solvents and do not require multiple tube transfers for most types of samples. The extraction of DNA from semen and very small bloodstains using Chelex 100 is as efficient or more efficient than using proteinase K and phenol-chloroform extraction. DNA extracted from bloodstains seems less prone to contain PCR inhibitors when prepared by this method. The Chelex method has been used with amplification and typing at the HLA DQ alpha locus to obtain the DQ alpha genotypes of many different types of samples, including whole blood, bloodstains, seminal stains, buccal swabs, hair and post-coital samples. The results of a concordance study are presented in which the DQ alpha genotypes of 84 samples prepared using Chelex or using conventional phenol-chloroform extraction are compared. The genotypes obtained using the two different extraction methods were identical for all samples tested.

Chelex 100 as a Medium for Simple Extraction of DNA for PCR-Based Typing from Forensic Material

https://ris.utwente.nl/ws/files/6782642/JIM1995.pdf

A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V.

Background DNA fragments that appeared to belong to an unidentified human herpesvirus were recently found in more than 90 percent of Kaposi's sarcoma lesions associated with the acquired immunodeficiency syndrome (AIDS). These fragments were also found in 6 of 39 tissue samples without Kaposi's sarcoma, including 3 malignant lymphomas, from patients with AIDS, but not in samples from patients without AIDS. Methods We examined the DNA of 193 lymphomas from 42 patients with AIDS and 151 patients who did not have AIDS. We searched the DNA for sequences of Kaposi's sarcoma–associated herpesvirus (KSHV) by Southern blot hybridization, the polymerase chain reaction (PCR), or both. The PCR products in the positive samples were sequenced and compared with the KSHV sequences in Kaposi's sarcoma tissues from patients with AIDS. Results KSHV sequences were identified in eight lymphomas in patients infected with the human immunodeficiency virus. All eight, and only these eight, were body-cavity–based lymphomas — that...

Kaposi's Sarcoma–Associated Herpesvirus-Like DNA Sequences in AIDS-Related Body-Cavity–Based Lymphomas

A very simple, fast, universally applicable and reproducible method to extract high quality megabase genomic DNA from different organisms is described. We applied the same method to extract high quality complex genomic DNA from different tissues (wheat, barley, potato, beans, pear and almond leaves as well as fungi, insects and shrimps' fresh tissue) without any modification. The method does not require expensive and environmentally hazardous reagents and equipment. It can be performed even in low technology laboratories. The amount of tissue required by this method is approximately 50-100 mg. The quantity and the quality of the DNA extracted by this method is high enough to perform hundreds of PCR-based reactions and also to be used in other DNA manipulation techniques such as restriction digestion, Southern blot and cloning.

Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques

One of the obstacles encountered when extracting DNA from a large number of samples is the cumbersome method of deprotein-izing cell digests with the hazardous organic solvents phenol and isochloroform. Several other non-toxic extraction procedures have been published, but require either extensive dialysis (1) or the use of filters (2). A rapid, safe and inexpensive method was developed to simplify the deprotein-ization procedure. This method involves salting out of the cellular proteins by dehydration and precipitation with a saturated NaCl solution. Buffy coats of nucleated cells obtained from anticoagulated blood (ACD or EDTA) were resuspended in 15 ml polypropylene centrifugation tubes with 3 ml of nuclei lysis buffer (10 mM Tris-HCl t 400 mM NaCl and 2 mM Na 2 EDTA, pH 8.2). The cell lysates were digested overnight at 37°C with 0.2 ml of 10Z SDS and 0.5 ml of a protease K solution (1 mg protease K in 1Z SDS and 2 mM Na2EDTA). After digestion was complete, 1 ml of saturated NaCl (approximately 6M) was added to each tube and shaken vigorously for 15 seconds, followed by centrifugation at 2500 rpm for 15 minutes. The precipitated protein pellet was left at the bottom of the tube and the supernatant containing the DNA was transferred to another 15 ml polypropylene tube. Exactly 2 volumes of room temperature absolute ethanol was added and the tubes inverted several times until the DNA precipitated. The precipitated DNA strands were removed with a plastic spatula or pipette and transferred to a 1.5 ml microcentrifuge tube containing 100-200 pi TE buffer (10 mM Tris-HCl, 0.2 mM Na 2 EDTA, pH 7.5). The DNA was allowed to dissolve 2 hours at 37°C before quantitating. The DNA obtained from this simple technique yielded quantities comparable to those obtained from phenol-chloroform extractions. The 260/280 ratios were consistently 1.8-2.0, demonstrating good deproteinization. Restrictions were performed using a number of different enzymes requiring high, medium or low salt concentrations, all resulting in complete restriction. This procedure has been used in our laboratory on several thousand blood samples for parentage, population and forensic studies. This technique is used with our non-isotopic hybridization procedures (3) rendering the entire process of RFLP analysis free of toxic materials.

A simple salting out procedure for extracting DNA from human nucleated cells

With a few exceptions DNA probing techniques require the use of radioisotopes and toxic DNA extraction techniques which render the method expensive, potentially hazardous and time-consuming. Most isotopic labeling techniques use the isotope 32P and require 3-10 days to visualize bands after hybridization. An alternative approach is based on the use of non-isotopic detection methods. The available non-isotopic techniques were assessed and their practicality tested. All probes analyzed were tested on samples extracted with a non-toxic extraction procedure using 6 M NaCl as the substitute for phenol and isochloroform. By manipulating probe sizes, blocking agents, selection of membrane and detection system, it is feasible to use non-isotopic labeling and detection in routine parentage testing. Reproducible results were obtained with labeling a variety of DNA probes of various sizes, plasmid and inserts. With an absence of waste disposal costs, probes that are stable for over two years and a staining procedure which takes 3-5 h versus days the technique is well suited for a normal laboratory setting. The next key to the acceptability of DNA testing will be the commercial availability of DNA probes for widespread use.

The use of biotinylated DNA probes in parentage testing: non-isotopic labeling and non-toxic extraction.

We have performed linkage analysis to determine the genetic relationship between the loci coding for coagulation factor XIII B (F13B) and the regulator of complement activation (RCA) gene cluster, comprised of the loci encoding C3b/C4b-receptor (CR1), C3d/Epstein-Barr virus-receptor (CR2), decay accelerating factor (DAF), C4b-binding protein (C4BP), and factor H (HF), as the products of these six loci show structural similarities. Here we report that the human F13B gene is also a member of RCA gene cluster and that it maps in close proximity to the gene encoding complement factor H. Coagulation factor XIII is a tetrameric complex composed of two A and two B polypeptides held together by noncovalent bonds (Schwartz et al. 1973, Chung et al. 1974). Factor XIII is activated by the serine protease thrombin which cleaves a 36 amino acid (aa) peptide from the amino terminus of the catalytic A subunit. In the presence of Ca 2+, the activated A' subunit is released from the B subunit and acquires the transglutaminase enzymatic activity which is responsible for the cross-linking of the fibrin monomers (reviewed in Folk and Finlayson 1977, Lorand et al. 1980). The precise function of the B subunit is not completely understood, but it is thought to protect or stabilize the A subunit, regulating the activation of the zymogen (Cooke 1974). The F13B locus displays a genetic polymorphism (Board 1980), and several phenotypic variants have been characterized by isoelectric focusing (IEF) (Dykes and Polesky 1987). Factor XIII B is structurally related to the members of the so-called superfamily of C3b/C4b binding proteins. These proteins have a structural organization based on the presence of internal repeat units of 60 aa which share a framework of highly conserved residues (reviewed in Reid et al. 1986). Factor XIII B is composed of ten of these repeats which represent the amino terminal 98 % of

Coagulation factor XIII B subunit is encoded by a gene linked to the regulator of complement activation (RCA) gene cluster in man

Human genomic DNA probes labeled by nick translation with biotin 11-UTP were successfully used to identify single copy restriction fragment length polymorphisms following electrophoresis and Southern blotting onto nylon membranes. Two human probes found on chromosome 21 were used, each of which demonstrated polymorphisms using two separate restriction endonucleases. Compared to isotopic labeling of probes the biotin labels are safer, faster and more economical. The detection method of streptavidin-biotin-alkaline phosphatase permitted phenotyping in a matter of hours after hybridization of the probe to the human DNA fragments.

The use of biotinylated DNA probes for detecting single copy human restriction fragment length polymorphisms separated by electrophoresis

Serum specimens from local Whites, Blacks and Amerindians were phenotyped for the B subunit of Factor XIII. Isoelectric focusing of desialylated samples on agarose gels at a pH 4–7 gradient followed by an immunoblotting technique was used for band identification. Gene frequencies for the three common alleles in Whites, F XIII*1, F XIIIB*2 and F XIIIB*3 were 0.776, 0.088 and 0.136, respectively, and were consistent with reports on this genetic marker system in European Whites. The Blacks and Amerindians were clearly differentiated from the Whites with F XIIIB*1,2 and 3 frequencies of 0.286, 0.635 and 0.079, respectively, for Blacks and 0.500, 0.034 and 0.466 for Amerindians. Based on the available population data on Factor XIIIB, this genetic system should prove to be a valuable marker for anthropological genetics and parentage testing.

Dale D. Dykes

Papers

A simple salting out procedure for extracting DNA from human nucleated cells

The use of biotinylated DNA probes in parentage testing: non-isotopic labeling and non-toxic extraction.

Coagulation factor XIII B subunit is encoded by a gene linked to the regulator of complement activation (RCA) gene cluster in man

The use of biotinylated DNA probes for detecting single copy human restriction fragment length polymorphisms separated by electrophoresis

Gene frequency distribution of the B subunit of factor XIII (F XIIIB) in Minnesota Whites, Blacks and Amerindians