Australian sabbatical visit: Notebook 1, Aug 1986-Feb 1987
Brownlee was attempting to set up an assay to detect point mutations in DNA by forming DNA-32P RNA heteroduplexes and subsequently detecting Watson-Crick mismatches with RNases. He was helped by Andy Choo at the host laboratory. Choo provided Brownlee with DNA of different individuals (experiment 3). Brownlee intended to probe for RFLPs in the factor IX gene, following up work of Peter Winship in Brownlee's lab in Oxford. Mismatches had been detected before in other genes (see experiment 4, Myers et al. 1985) but Brownlee hoped to improve on Myers by using other RNases or different conditions of RNase digestion.
- Normal DNA individual samples from Andy Choo (experiment 3) for probing Factor IX gene for Taq I RFLP; results (experiment 7) on p. 26, either 1.8 or 1.3 or 1.3/1.8 heterozygote.
- Exon f, Factor IX DNA cloned into SP64 or SP65 from Peter Winship (experiment 10), subsequently shown to be suspect (experiment 14). Cloning of 470 nt long exon f segment of factor IX in pGEM riboprobe plasmids (experiment 18) and their transcription (experiments 20, 21).
- P- RNase of heteroduplexes – series of experiments (e.g. experiment 42) showing some evidence of specific RNase cleavage, but not at expected sites. Decided to make a shorter 32P RNA probe (experiment 52, 53) to aid identification of mismatches (continued in Australian Notebook 2, below).
- (With Ruth Brown) decided to construct a cosmid library, partial Sau 3A in pDVCos A2 (see experiments 38, 45 etc.) of one of her human-mouse hybrids containing a translocation presumed to be at or close to the human X-linked fragile X locus, that caused fragile X linked mental retardation (locus had not been cloned then).
- Details of somatic cell hybrid translocated at or close to fragile site (experiment 30 and insert).
- Aug 1986-Feb 1987