Determination of Unknown Genomic Sequences Without Cloning

The inherent problems of sensitivity and specificity that one encounters when trying to determine a particular nucleotide sequence directly in its genomic context can be overcome by selective amplification of the region of interest. This amplification of the target DNA is usually achieved by one of two strategies: The relevant piece of DNA may be cloned and therefore amplified in a bacterial cell or, alternatively, the desired fragment may be amplified in vitro using PCR technology. Both strategies have drawbacks. The cloning of a specific genomic sequence is labor-intensive, lengthy, and sometimes even difficult to achieve. The PCR amplification requires that enough sequence information is known to be able to design the two specific amplification primers and is therefore limited to sequencing alleles of already known DNA. There are, however, many cases that would benefit from the determination of unknown genomic sequence close to a known piece of DNA. With a particular cDNA in hand, one may wish, for example, to determine genomic gene sequences, such as the promoter of the gene, its introns, or 5′- and 3′-non-transcribed regions. The protocol presented here uses ligation-mediated PCR (LM-PCR) to amplify unknown genomic DNA next to a short stretch (about 100 bp) of known sequence and details a convenient procedure to determine the new sequence by dideoxy sequencing (1 ) . The procedure may form the basis for “walking sequencing” strategies in order to determine large regions of continuous sequence information starting from a limited piece of known DNA.