The aim of this project is to develop methods which will allow one to select chromosome specific and tissue specific cDNA clones. Chromosome 21 will be used as a target, and cerebellum will be the source of the RNA for this project. These choices are based on the fact that cerebellum tissues express a very high number of mRNAs at low frequency, and on the potential usefulness of any isolated sequences in the study of trisomy 21. However the technical problems involved are general and, if solved, would lead to methods of general utility.
The research was predicated on 2 widely held and frequently reported aspects of polymerase chain reaction (PCR): the products of multiple cycles of PCT do not accurately reflect the starting amounts and after a finite number of cycles the PCR reaches a plateau. Following 35 cycles of PCR an equivalent amount of product was obtained from a 1000-fold range of unique template deoxyribonucleic acid (DNA). PCR is therefore not quantitative in this case. However, when 2 targets of slightly different size were used as templates, the DNA which was present at the lower initial concentration was not amplified once the plateau was reached by the predominant template. The window for equivalent amplification was very narrow. This puts a cap on the degree to which normalization can occur by this method.
To see if the approach was more fruitful with starting material of greater complexity, chromosomal deoxyribonucleic acid (cDNA) libraries were prepared with or without a PCR normalization step using human uterus, cervix, brain or ovary cell ribonucleic acid (RNA). The results indicate that the PCR based method is not going to yield normalized libraries as had been hoped.
RNA from human erythroleukemia or a glioblastoma astroblastoma cell line was used in CoT hybridization experiments to achieve the same goal. The results show that the rare cDNAs were increased approximately 3.5-fold relative to medium sequences or 36-fold in relation to high level sequences. This is not an adequate degree of normalization.
To achieve an enrichment of chromosome 21 sequences, experiments were performed in which the human Glud, b-globin and actin genes were immobilized on cellulose matrices, sepharose F/F matrices and nylon matrices. After 2 cycles of selection, libraries were prepared and screened for enrichment of 3 chromosome 21 sequences. A very minimal enrichment was achieved.
To achieve the above goals one could use the standard approach of cDNA bank construction, differential hybridization and use of the selected sequences as probes to Southern blots from cell lines which contain different chromosome fragments to define their origin. In the more streamlined approach which is proposed in this project, the cDNA population generated from the cerebellum cell RNA will be amplified using the Polymerise Chain Reaction (PCR) method with a final asymmetric step or T7 polymerase transcription to generate amplified single strand cDNA or RNA.
By using a high number of cycles the usual disadvantage of the nonquantitative nature of PCR amplification can become advantageous by diminishing the differences between frequent and infrequent sequences in the population of the cDNA. An additional normalization step through a more standard approach (ie back-hybridization followed by hydroxyapatite fractionation) would further decrease the differences in the abundance among the various transcripts in the cDNA population. Irrespective of the methods used to prepare the seminormalized cDNA, the next step will aim to restrict the cDNA to those sequences that are transcribed from chromosome 21. This will involve the hybridization of the single strand cDNA or RNA transcribed by T7 polymerise to immobilized chromosome 21 DNA. This will be prepared from a chromosome specific library. The retained single strand cDNA or RNA will then be converted into double strand DNA prior to the generation of the chromosome specific library. This will be of use in its own right.
The final step will involve differential hybridization. For this a second cDNA library will be prepared using human uterus (or other tissue) mRNA as the starting material. The probes for the differential hybridization will again be generated by a combination of cDNA and PCR technology. This should allow positive signals to be obtained from minor sequences whereas standard cDNA probe methods are unlikely to detect sequences which are present at less than 1 part per 1000. Clones which are selected by differential hybridization will be tested subsequently for their tissue and chromosome specificity.
The overall procedures in the project would provide sequences for many different uses in the Human Genome Analysis Programme and should result, by the combination of PCR amplification, T7 polymerase transcription and hybrid selection, in cDNA banks which are normalized by one rapid protocol. In parallel, a more standard Cot-limited hybridization will be used to provide a comparison with these more innovative approaches.
Funding SchemeCSC - Cost-sharing contracts