Ligation-mediated PCR for genomic sequencing and footprinting.
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.
Mueller
P R
PR
California Institute of Technology, Pasadena, California, USA.
Wold
B
B
Garrity
P A
PA
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
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