A procedure forin vitroamplification of DNA segments that lie outside the boundaries of known sequences

Abstract

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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Structure and organization of amplicons containing the E4 esterase genes responsible for insecticide resistance in the aphid Myzus persicae (Sulzer)

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A system for insertional mutagenesis and chromosomal rearrangement using the Ds transposon and Cre—lox

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