We describe a technique for transferring electrophoretically separated bands of RNA from an agarose gel to paper strip The RNA is coupled covalently to diazoenzylomethyl groups on the paper. After transTer and appropriate treatment of the paper to destroy remaining diazo goups, specific RNA bands can be detected by hybridization with mP~labeled DNA probes followed by autoradiography. This procedure allows detection of specific RNA ban s wit high sensitivity and low background. was allowed to crystallize. It was collected on a sintered glass filter, washed with pyridine, then washed thoroughly with petroleum ether and dried under reduced pressure. The NBPC (267 g) was stored at -20°in a desiccator.Preparation of Aminobenzyloxymethyl-Paper. The method for making aminobenzyloxymethyl (ABM)-paper and its subsequent conversion to the diazobenzyloxymethyl form (DBM-paper) by diazotization is outlined in Fig. 1. The ABM-paper was prepared by a modification of methods described previously (5-7) for preparing aminobenzyloxymethyl-cellulose powder. A sheet of Whatman 540 paper (14 X 25 cm) in a flat enameled pan was soaked with 10 ml of an aqueous solution of 0.8 g of NBPC and 0.25 g of sodium acetate. Air bubbles under the paper were squeezed out and the paper was dried at 600 and then heated to 130-135°for 35 min. The paper was washed twice for 20 min with water, dried at 600, washed twice for 20 mm with benzene, and dried in the air. The nitrobenzyloxymethyl paper was reduced to ABM-paper by treating it with 150 ml of 20% sodium dithionite (wt/vol) for 30 min at 600 with shaking. The ABM-paper was washed for 20 min with water, 20 min with 30% acetic acid, and then with water until there was no further odor of H2S. The ABM-paper was dried in the air and stored at 40 in a desiccator. It is stable for several weeks under these conditions. Diazotization of ABM-Paper. Just before reaction with single-stranded nucleic acids, ABM-paper was converted to the diazobenzyloxymethyl (DBM) form by treatment with a solution containing 40 ml of water, 80 ml of 1.8 M HCO, and 3.2 ml of a freshly prepared solution of NaNO2 (10 mg/ml) for 30 min at 40. The solution was checked for free HNO2 with starchiodide paper, which turns black. After 30 min, the DBM-paper was washed five times for 5 min each with 100 ml of cold water and then twice for 10 min with ice-cold sodium borate buffer, 50 mM, pH 8. Upon washing, the paper turns bright yellow. It should be kept cold until transfers begin, no more than 15 min later. DBM-paper had the capacity to couple [16][17][18][19][20][21][22][23][24]

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The polymerase chain reaction (PCR) system allows the amplification of DNA segments between two regions of known sequence [11. We describe a procedure that extends this technique to sequences that lie outside the boundaries of known sequences. The approach simply requires inversion of the sequence of interest by circularizaton and reopening at a different site. Figure 1 shows the procedure for amplifying segments (X and Z) in a genome, adjacent to a segment (Y-Y') of known sequence. The genome is first cleaved with any restricton endonuclease (N and N' in Fig. 1) with convenienfly located sites. The diluted DNA (< 3 gml) is then ligated ovemight. The circles are then cleaved at any site located between Y and Y. Regions X and Z can be amplified using oligonucleofide primers corresponding to regions Y and Y', synthesized in opposite orientation to that for normal PCR because they have been inverted and now flank regions X and Z (Fig. 1). We therefore designate the approach inverted PCRO (IPCR). The point at which the two ends were ligated is defined by the site N. To test this procedure we used the gene encoding the precursor to the major merozoite surface antigens of P.falciparum 121. Chromosomal DNA (2igg) was cut with Rsal, ligated, re-cut with Hinfl and amplified. The expected 297 bp fragment was obtained (Fig2A&B) and checked by sequencing nine clones. It is not necessary to cleave the circles with restricton endonuclease M as the same effect can be achieved by Introducing nicks by heafing (Fg.2C). Many other variations of IPCR could obviously be employed. Sites could be included in the oligonucleotides to facilitate cloning. Instead of a single enzyme, two different enzymes could be used, with end-filling if necessary. Instead of cleavage with N, the DNA could be randomly

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