Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.796654
Title: Optimization and application of chromosome in situ suppression hybridization
Author: Isa, Mohamed Nizam H. J.
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1991
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Abstract:
The overall aim of this project was to develop the technique of chromosomal in situ suppression (CISS) hybridization using whole chromosome specific libraries (chromosome painting) and to apply it to the investigation of diagnostic problems in clinical cytogenetics. Initially to gain experience with non-isotopic in situ hybridization, repetitive target probes DYS59 (GMY10) and DYS58 (GMGY7) were used. This provided experience in labelling of probe with biotin, hybridization and detection conditions (alkaline phosphatase detection) and analysis of results. The technique was reliable and sensitive and was applied to map the gene for angiotensinogen to 1q42. In the later part of this initial work, a fluorescence detection technique using fluoresceinated avidin and goat biotinylated anti-avidin was applied to confirm an isochromosomes Yp and Yq using DYS59 (GMGY10) and DYS58 (GMGY7) probes. The study then progressed into the development of the chromosome painting technique. Difficulties were encountered in preparing the working library probe from the chromosome 21 specific library and a major part of the work involved solving these problems. The libraries were found to be less concentrated than indicated by the supplier. Consequently, the amplification and purification following established protocols failed to produce a concentrated DNA library in the phage. However, a good yield of the DNA library was achieved by using trypticase in the culture media and high purity agarose as the top agar during the amplification. Labelling of the library by nick-translation and random priming did not achieve decoration of the whole chromosome 21 but direct labelling of Biotin-ll-dUTP by polymerase chain reaction (PCR) amplification was found to be efficient and overcame the problem of non-homogenous painting of the target chromosome. This direct labelling approach had difficulties in the cleaning and concentration of the PCR product. These were overcome by cleaning with Sephadex G-50 column chromatography and freeze drying of eluate. Once homogeneous painting had been achieved the probe was applied for chromosome painting. Many problems and parameters for the optimum working conditions were identified in this part of study. These are either independent or/and related to various conditions involved during all stages of the technique. The maximum final concentration of the DNA mixture per slide was 10ug/10ul and increasing the ratio of the probe and/or the unlabelled DNA did not improve either the quality of suppression or the hybridization signal. Addition of human cot 1-DNA in 1 to 4 ratio with total human DNA gave better suppression. Denaturation of labelled probe and competitor DNA mixture was optimum at 75C for 8 minutes and for optimum preannealling, the mixture was prehybridized for a minimum of 60 minutes at 37C. Slides were only treated with RNase when necessary and not with Proteinase K as the latter tended to wash the cells off the slide. Denaturation of the slides was carried out at 70-75C in 70% formamide/2XSSC for a maximum of 8 minutes. At temperature of 80C the chromosome morphology was found to be distorted. Hybridization when carried out at 37C for 15 to 20 hours showed good hybridization with chromosome morphology undisturbed. Hybridization at 42-45C showed crystallization and heavy background deposits. Posthybridization washing in three changes of 50% formamide/2XSSC at 45C was found to be optimal in producing a clean background. In between detection washing using 0.1M sodium phosphate buffer with 0.1% Nonidet P-40 carried out at room temperature is sufficient to remove excess stain as compared to other washing buffers such as 2XSSC or 4XSSC containing Triton-X or Tween 20. Detection was carried out at room temperature for 15-20 minutes and any slide dried during this stage produced high autofluorescence of fluoresceinated avidin which was difficult to remove by washing. A single amplification cycle was sufficient to enhance the decoration of chromosome 21. Prebanding of slides prior to hybridization did not affect the target chromosomes, however, incomplete destaining did hinder probe penetration and interfere with counterstaining. It was found that refixing of slides (either new or old slides) in methanol:acetic acid (3:1) before denaturation tended to improve the hybridization result as well as reducing background signal. In general, the technical difficulties were related to either probe preparation, poor hybridization, non-homogeneous painting or high background but with modifications of the parameters as detailed above the method was shown to be reliable and reproducible. Chromosomes obtained from phytohaemagglutinin (PHA) stimulated blood cultures were used during the initial phase. Subsequently, painting was successfully performed on cytogenetically normal metaphase and prometaphase samples of cultured amniocytes, lymphoblastoid cell lines, chorionic villus samples (CVS) and bone marrow preparations. The results showed that all normal chromosome 21s in all types of preparation except direct chorionic villus sample (CVS) were intensely painted and distinctly recognisable. However, results with interphase nuclei were not encouraging. The signals produced were not consistent enough to produce as reliable results. Twelve cases with cytogenetic abnormalities involving the chromosome 21 were investigated using chromosome painting. These results proved that chromosome painting can be used for rapid identification of individual chromosomes and is complementary and confirmatory to conventional karyotyping and as such is predicted to have a future routine diagnostic role in clinical cytogenetics in additions to its research applications.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.796654  DOI: Not available
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