| 摘要 |
<p>Preparing DNA probes (I) with single-stranded DNA oligomers (II) of a repetitive sequence motif. Preparing DNA probes (I) with single-stranded DNA oligomers (II) of a repetitive sequence motif comprises: (a) preparing (II) as sense strand; (b) addition of antisense strands, complementary to (II) and allowing hybridization to occur; (c) adding a first primer (P1), comprising a priming sequence (PS) and an oligomer of the repetitive sequence and a second primer (P2) comprising a PS and oligomer complementary to the sense strand, and allowing these to hybridize; (d) adding nucleotides (nt), Klenow fragment and a ligase, and reacting; (e) removing any single-stranded DNA and any DNA sequence shorter than 100 nt, by usual methods; (f) amplification of the purified DNA by PCR; and (g) labeling the PCR products with a reporter molecule. Independent claims are also included for: (1) a variant of the new method in which P1 and P2 are added to separate batches of the initial hybridization mixture; allowing them to hybridize; performing steps (d) and (e) on each mixture; combining the batches; denaturing DNA and renaturing; then performing steps (f) and (g); and (2) kit for the new process comprising single-stranded DNA oligomers (sense and antisense), primers, nt and the specified enzymes. Preferred Process: The oligomers of the sense and antisense strands have the same length, and the two priming sequences are identical. The repeated sequence motif is particularly the human telomer repeat and the reporter is either a fluorophore or a radioisotope. The major product of step (b) is double-stranded DNA with pairing gaps in both strands and with sticky ends. The primer hybridizes to the 3'-sticky ends; gaps are filled in (Klenow) and then phosphodiester bonds formed (ligase). Preferred Materials: The sense and antisense oligomers for the human telomer are 3'-(GGGATT)5 and 5'-(CCCTAA)5 and the primer is 5'-CGATCGCTCGAGGAATT.</p> |
