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You want to know the sequence of a gene and only know the sequence of 2 DNA markers near it such as an RFLP (stands for Restriction fragment length polymorph) or another gene. Using different restriction enzymes, cut the genome of the organism to create DNA clones which overlap. For example, if the genome is ABCDEFGHI, enzyme 1 cuts it into 2 pieces to make ABCD EFGHI and enzyme 2 cuts it into 3 pieces to make ABC DEF GHI.. These pieces will over lap in sequence because the enzymes have different restriction sites.
Using the known sequence of the RFLP, a radio labeled nucleic acid probe can be made to search the genomic library. The probe will anneal to clones with overlaps, and the ends of these clones can be made into new probes to search for the adjacent sequence. This is repeated until the desired segment has been cloned. each clone is sequenced and the overlaps are compared to determine the order of the sequences.
This technique can be used to analysis diseases, such as cystic fibrosis, to look for mutations.
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