![]() ![]() The history of the development of DNA blotting is described in this chapter. There was no evidence for a role for HPV in the aetiology of skin cancer in RARs in south-eastern Queensland but the possibility remains that as yet unidentified HPV types are involved. The incidence of HPV 5 (or 5-related HPVs) in RAR SCC was very low and that of HPV DNA in RAR warts was lower than that recorded elsewhere but this was not due to insensitivity of the assays. HPV DNA was identified in another 5 skin biopsies from another 4 RARs: HPV 3A in a wart and a hyperkeratosis, HPV 3/10-related DNA in 2 solar keratoses and HPV 5/8-related DNA in another (20-50 copies/cell). Only HPV 1A in the SCC exhibited a significant degree of subtype variation. Genomic HPV DNA was characterised in 7 skin biopsies from 4 renal allograft recipients (RARs): HPV 1A in a SCC (20 copies/cell) and a BCC (10 copies/cell) from the one patient, HPV 36 (20 copies/cell) in a SCC, HPV 1A 1000 copies/cell) in a wart and HPV 2B (200-800 copies/cell) in 3 warts from the one patient. ![]() Human papillomavirus (HPV) DNA was identified by Southern blot hybridisation using, as individual probes, purified insert DNA from recombinant HPV 1, 2, 3 or 3/10, 4, 5 or 5/8, 7, 11, 16, 18 and 41 under relaxed conditions and characterised by restriction enzyme analysis and Southern blot hybridisation under more stringent conditions. The 104 skin biopsies from 34 patients who attended a Renal Transplant Unit in Brisbane over 12 months included 40 squamous cell carcinoma (SCC), 22 solar keratoses, 4 hyperkeratoses, 18 warts and 11 basal cell carcinoma (BCC). Trenfield, K Salmond, C A Pope, J H Hardie, I R Southern blot analysis of skin biopsies for human papillomavirus DNA: renal allograft recipients in south-eastern Queensland. Recent developments of hybridization membranes and buffers have resulted in increased sensitivity closing. Northern blotting is relatively simple to perform, inexpensive, and not plagued by artefacts. is analysed by hybridization to one or more specific probes that are labelled for subsequent detection. In short, the RNA is size-fractionated by gel electrophoresis and transferred by blotting onto a membrane to which the RNA is covalently bound. ![]() Northern blotting analysis is a classical method for analysis of the size and steady-state level of a specific RNA in a complex sample. The downward capillary method described in This slows down the blotting process and may reduce the amount of DNA that can be transferred. The traditional method of upward capillary transfer of DNA from gel to membrane described in the first basic and alternate protocols has certain disadvantages, notably the fact that the gel can become crushed by the weighted filter papers and paper towels that are laid on top of it. A second alternate protocol describes a transfer method based on a different transfer-stack setup. The method can also be used with neutral nylon membranes but less DNA will be retained. The advantage of this combination is that no post-transfer immobilization step is required, as the positively charged membrane binds DNA irreversibly under alkaline transfer conditions. An alternate protocol details transfer using nylon membranes and an alkaline buffer, and is primarily used with positively charged nylon membranes. A Support Protocol describes how to calibrate a UV transilluminator for optimal UV irradiation of a nylon membrane. ![]() Immobilization is achieved by UV irradiation (for nylon) or baking (for nitrocellulose). With the high-salt buffer, the DNA becomes bound to the membrane during transfer but not permanently immobilized. This appendix describes Southern blotting via upward capillary transfer of DNA from an agarose gel onto a nylon or nitrocellulose membrane, using a high-salt transfer buffer to promote binding of DNA to the membrane. After immobilization, the DNA can be subjected to hybridization analysis, enabling bands with sequence similarity to a labeled probe to be identified. Southern blotting is the transfer of DNA fragments from an electrophoresis gel to a membrane support (the properties and advantages of the different types of membrane, transfer buffer, and transfer method are discussed in detail), resulting in immobilization of the DNA fragments, so the membrane carries a semipermanent reproduction of the banding pattern of the gel. DNA was appropriate for Southern blotting when the tissue was obtained less than three to five. Histological examination showing slight or no autolysis and the presence of ribosomal bands after gel electrophoresis were both indicative parameters of RNA preservation. Was obtained less than two days postmortem. Northern and Southern blot analysis of human RNA and DNA in autopsy material ![]()
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