title: Ligation-mediated PCR for genomic sequencing and footprinting.
abstract: This unit describes how PCR can be used to exponentially amplify segments of DNA located between two specified primer hybridization sites. A single-sided PCR method is used that initially requires specification of only one primer hybridization site; the second is defined by the ligation-based addition of a unique DNA linker. This linker, together with the flanking gene-specific primer, allows exponential amplification of any fragment of DNA. Because a defined, discrete-length sequence is added to every fragment, complex populations of DNA such as sequence ladders can be amplified intact with retention of single-base resolution. The ligation-based protocol was specifically designed for genomic footprinting and direct sequencing reactions, and is described in this context; it can, however, be used for other applications.
authors.affiliation: California Institute of Technology, Pasadena, California, USA.
authors.forename: P R
authors.initials: PR
authors.lastname: Mueller
authors.forename: B
authors.initials: B
authors.lastname: Wold
authors.forename: P A
authors.initials: PA
authors.lastname: Garrity
mesh_terms: D002478:Cells, Cultured / Q000737:chemistry; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D018983:DNA Footprinting / Q000379:methods*; D011088:DNA Ligases; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*; D013048:Specimen Handling / Q000379:methods; D013463:Sulfuric Acid Esters; D013535:Suspensions
chemical_list: D017931:DNA Primers; D013463:Sulfuric Acid Esters; D013535:Suspensions; D004247:DNA; C070331:Tli polymerase; D004259:DNA-Directed DNA Polymerase; D011088:DNA Ligases; C007482:dimethyl sulfate