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Unknown Genomic Sequences 373
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Determination of Unknown Genomic Sequences
Without Cloning
Jean-Pierre Quivy and Peter B. Becker
1. Introduction
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′-nontranscribed regions. The protocol presented
here uses ligation-mediated polymerase chain reaction (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 to determine
large regions of continuous sequence information starting from a limited piece of
known DNA.
The central feature of the LM-PCR technique is the ligation of a known short
oligonucleotide, the “linker,” to selected ends of genomic DNA fragments (Fig. 1).
These generic linker sequences provide the second primer for amplification of linked
fragments in combination with an oligonucleotide based on known sequences. LM-PCR
was first introduced for genomic footprinting and chemical sequencing (2,3). The
disadvantages of chemical sequencing over the chain termination method (1) prompted
us to adapt LM-PCR technology for direct dideoxy sequencing of genomic DNA (4).
The underlying procedure is derived from a variation of the original LM-PCR protocol
called “Linker Tag Selection LM-PCR” (5).
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
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