Chromosome Painting Using Microdissection Techniques

Genetics

Stefanou, Eunice-Georgia G

University:University of Glasgow

关键词: Genetics;   

Others  :  http://theses.gla.ac.uk/75497/1/13832093.pdf

来源: University of Glasgow

【 摘 要 】

Chromosome microdissection is a technique that was first described in 1981 by Scalenge et al. Since then, several modifications have been made and it was recently applied to dissect abnormal parts of aberrant chromosomes, the origin of which was unknown, with the ultimate aim of identifying the origin of the dissected chromosomal material (Meltzer et al. 1992). The aim of this project was to develop the technique, so that it could be further used as a molecular cytogenetic tool in the delineation of chromosome aberrations encountered in the diagnostic workload of this department which remain unidentifiable by using standard cytogenetic techniques. The steps associated with the technique involved the microdissection of several copies of a specific chromosomal region, PCR amplification using a degenerate oligonucleotide primer (DOP primer with six 'N' degenerate bases), labelling of the amplified product with biotin-11-dUTP and finally, reverse chromosome painting by hybridising the labelled product on normal metaphase cells. The location of the hybridisation signal would then reveal the chromosomal origin of the microdissected DNA of interest. All parameters that could affect the efficiency of the DOP-PCR amplification, such as the primer concentration and annealing temperatures were evaluated by using low concentrations of at first, human genomic DNA and finally FACS isolated copies of chromosome 4 as a positive control. Satisfactory results meant that the values of the parameters tested were kept constant for all further attempts. Contamination was the main drawback in interpreting the PCR results. The nature of the DOP primer that could amplify literally any type of DNA template, in conjunction with the fact that the starting microdissected material did not exceed in total a few hundred femptograms (fg) (an average size band equals to 15-50fg, 1fg=10-9 mug) made the amplification extremely sensitive to extraneous contaminations. It was therefore necessary to sequentially introduce multiple specificprecautions (i.e. barrier-pipette tips, autoclaving all solutions, preparing small aliquots of each of the PCR reagents to allow only a single use per experiment and performing all steps involved with the PCR inside a sterile safety cabinet) in order to prevent contamination. Several approaches to chromosome microdissection were investigated. Successfiil results were obtained by dissecting the fragment of interest and transferring it into a collection tube by gently touching the tip of the needle inside the collection drop, so as to release the DNA fragment without having to break the tip of the needle inside the tube. For this, it was very important to obtain the correct size of needles. Successfiil micro-FISH probes were produced by less than ten fragments, proving the efficiency of the technique. Successful results were obtained from microdissections of the chromosome regions 7p21-31, 21q21-22, 16cen, 16cen→q22 as well as double minutes present in an abnormal Acute Myeloid Leukaemia case (AML). Signals produced by FISH were very bright, covering fully the corresponding region. Using competitor DNA totally eliminated cross-reactivity with the other chromosomes of the cells. The successful micro-FISH results indicated that the conditions of all steps involved were appropriate for further generation of micro-FISH probes, providing that contamination was either absent or present at very low levels at the first critical round of amplification. These results suggest that chromosome microdissection should have the potential for introduction into routine clinical practice. The next stage is to use the optimised approach described here in an unselected series of cytogenetic cases to see if its potential as a diagnostic tool can be realised.