Gene analysis using oligonucleotides (1989)

A method has been developed utilising the specific hybridisation properties of oligonucleotides, to detect single base changes in DNA using a sandwich-assay.The method is based on the covalent imnobilisation of one oligonucleotide (capture probe) to a solid support resin and the use of a second 32 P labelled oligonucleotide (detector probe) free in solution.In the presence of complementary target D NA both probes will hybridise, resulting in the resin being radioactively labelled.When exposed to stringent hybridisation and washing conditions,only an exactly matched detector probe will remain bound to the target DNA.This relies on the property that mismatched sequences are destabilising to the hybrid complex and will demonstrate a reduced melting temperature under stringent conditions.The genotype of the target DNA can thus be established by running two assays in parallel,with labeled oligonucleotide probes specific for the normal and the nutated coding sequence.Using the sicikle cell condition of the beta-gldbin gene as a model system, a sandwich assay has been optimised for the genotypic analysis of small ( < 200 ng) samples of human genomic DNA.Under optimum hybridisation conditions a detection limit of 1 femtomoles target DNA was found.The analysis of single copy genes (attcmolar concentrations of target DNA) using this assay was possible with the application of the polymerase chain reaction.Preliminary work has been carried out to adapt this assay for the detection of two frequently occurring base mutations associated with the alpha-l-antitrypsin gene and correlated with the Z and S Pi types.Efforts have also been made to adapt this assay for a non-radioactive labeling strategy.The sandwich assay was tested on clinical blood samples from patients of all three genotypes to the sidkle condition.Disregarding ABREVIATIQWS A/C/G/T dA/dC/dG,dT,dU 2 -aminoadenine alpha-1antitrypsin Adenine, cytosine / guanine, thymidine Adenosine, cytidine, guanosine, thymidine, uridine deoxyribonucleotide 1 (2 / 6-diaminopur ine / D ) o<-1-antitrypsin (MZ/Z alleles and MS/S alleles) AIDS AMP ATP dATP/dCTP dGTP/dTTp I n t r o d u c t i c n Advances in molecular biology and biotechnology have lead to many exciting possibilities for DNA diagnosis.The developments have altered many aspects of m o d e m medicine including the pre-and postnatal analysis of genetic disease,also the analysis of infectious and acquired diseases (for example,malignancies) .These developments have, to a large part,been stimulated by the increasing awareness of infectious disorders,and the demand for carrier detection of many inherited diseases.Only very recently,however,are clinical laboratories considering DNA probes as routine diagnostic reagents.This seems surprising when one considers that the potential applications of the ENA probe were elucidated more than 30 years ago with the discovery of the double helical structure of nucleic acids by Watson and Crick.The main reasons for this delay in accepting DNA probes as useful tools have been the limited availability of non-radioactive techniques, the poor methods of sample preparation, the lack of experience with "dirty" clinical samples and a lack of automation.For ENA diagnostics to be taken up on a routine basis,methods have to be developed which are simple,inexpensive, sensitive, specific in detection,and reliable.Furthermore,any method developed needs to be a technique adaptable for the diagnosis of any other newly found defects.Oligonucleotides have established themselves as useful tools for the detection of single base mutations .Their properties provide a new means of diagnostic accuracy that relies on the use of DNA sequence information .The development of detection methods using oligonucleotides will not only open up the possibility of analysing many inherited disorders,but may also further other developments such * H o r s t et a l .(19 8 6 ) * T hein et a l .(1 9 8 6 ) * G o o s s e n et al. (1 9 8 2 ) * Collins et al.(1 9 8 4 )x Dilella et al. (1986) Antithrombin III deficiency Cancer Antithrcmbin