ABSTRACT
We have developed a semi-automated HLA class I typing system utilising TET/TAMRA-labelled fluorescence resonance energy transfer (FRET) hydrolysis probes. The results from 87 individuals are in full concordance with serology and conventional gel-based systems. This assay replaces labour-intensive conventional gel-based DNA typing and has a higher allelic resolution than serology. Our approach differs from previously published fluorogenic probe typing protocols in that it provides simultaneous typing of HLA-A, -B and -C loci to medium resolution. Furthermore, by using equipment that is not specific to FRET probe analysis our system has in-built expansion capacity to 384 reactions per plate, thus making it applicable to high-throughput population screening. Automation is achieved through the use of computer software which analyses direct input from the fluorescence reader, allowing high throughput with a low inherent error rate.
Subject(s)
Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/immunology , Histocompatibility Testing/methods , DNA Primers , Fluorescent Dyes , Humans , Polymerase Chain Reaction/methods , Sensitivity and Specificity , Spectrometry, FluorescenceSubject(s)
Antibodies, Antinuclear/immunology , Antibodies, Monoclonal , DNA/isolation & purification , Immunosorbent Techniques , Animals , Cells, Cultured , Electrophoresis, Agar Gel , Humans , Hybrid Cells , Lupus Erythematosus, Systemic/immunology , Magnetics , Mice , Mice, Inbred Strains , Microspheres , Oligodeoxyribonucleotides/metabolism , Polymerase Chain Reaction , SpleenABSTRACT
Extraction of DNA from old skeletal material is of great importance in the identification of human remains, but is particularly difficult because the methods currently employed, especially those using phenol/chloroform, are not always satisfactory. A simple technique based on the removal of non-nucleic acid material by salting out (precipitation) with saturated sodium acetate is described; the presence of DNA in the extract being confirmed by amplification of selected sequences of the HLA-DRB1 gene using the polymerase chain reaction (PCR). The method was applied to fresh bone (five femoral heads and six vertebral bodies) and to bone from two forensic cases, 3 and 9 months post-mortem, respectively. Parallel extractions using the phenol/chloroform technique were performed on all samples in order to compare the efficiency of the two methods. Using sodium acetate precipitation, amplifiable DNA was consistently extracted from fresh bone, as well as from the two forensic cases. With the phenol/chloroform method, amplification was successful in only 7 out of 11 instances with the fresh bone samples and failed in both forensic cases. The studies also showed that an effective way of removing PCR inhibitors is to subject the extract to agarose gel electrophoresis, isolate the high molecular weight area and re-extract the DNA from the gel by boiling. It was concluded that the sodium acetate method is a valid alternative to established techniques for extracting DNA from bone and that it offers the advantages of being simple, quick, inexpensive and avoids using hazardous reagents.
