Ligation-mediated PCR amplification of specific fragments from a Class-II restriction endonuclease total digest

Abstract

A method is described which permits the ligation- mediated PCR amplification of specific fragments from a Class-II restriction endonuclease total digest. Feasibility was tested using Bcl I and phage lambda DNA as a model enzyme and amplicon system, respectively. Bcl I is one of many widely used restriction enzymes which cleave at palindromic recognition sequences and leave 5'-protruding ends of defined sequence. Using a single pair of universal primers, a given fragment can be specifically amplified after joining the fragments to adaptors consisting of a duplex primer region and a 9-nucleotide protruding single-stranded 5'-end containing the sequence complementary to the cleaved restriction site and a 4-nucleotide 'indexing sequence.' The protruding strand anneals to a restriction fragment by displacing its corresponding strand in the same fragment-specific indexing sequence located juxtaposed to the restriction site. The adaptor is covalently linked to the restriction fragment by T4 DNA ligase, and amplification is carried out under conditions for long-distance PCR using the M13 forward and reverse primers. The technique discriminated robustly between mismatches and perfect matches for the 16 indexing sequences tested to allow individual lambda Bcl I fragments to be amplified from their respective adaptor pairs. A strategy is proposed enabling a non-cloning approach to the accession, physical mapping and sequencing of genomic DNA. The method could also have application in high-throughput genetic mapping and fingerprinting and should expand the enzyme base for ligation- mediated indexing technology which has previously been limited to the Class-IIS and IP restriction endonucleases.

Adaptor Long-Range PCR Procedure for Clone-Specific Characterization and Chromosomal Localization

“…In summary, the powerfulness of ALR-PCR is anticipated to be a combination of the following parameters: (i) ready-to-use polymerase mixtures and a master mixture, which reduce time, experimental steps, and the requirements of PCR optimization for each polymerase mixture (3,4); (ii) a 5-min ligation procedure; (iii) rapid purification of DNA digests; (iv) optimized DNA template concentration protocol to avoid nonspecific amplification and high backgrounds; (v) long-range PCR protocol to obtain at least 9.6 kb single PCR products; similar work but with different conditions (17) has emphasized the problems associated with PCR-amplifying large PCR products; (vi) two-step PCR cycling with the same annealing and extension temperature at 68C, resulting in high-stringency amplification as previously described (18,19) and also applied in our work; (vii) simple design of the adaptors according to the preferred restriction endonuclease enzyme; and (viii) simple technology and equipment required. ALR-PCR therefore offers a more simple, flexible, and user-friendly approach over other adaptor-PCR procedures.…”

Measuring and monitoring apoptosis and drug toxicity in HIV patients by ligation‐mediated polymerase chain reaction

“…Though LM-PCR exists in various guises [18,40,41], we have developed one form of LM-PCR into a new apoptosis quantifier, showing it to be reproducible and at least as sensitive as other accepted apoptosis quantifiers. This work is important because LM-PCR can assist in monitoring disease processes and in understanding the role of apoptosis in certain pathologies.…”

LAMPS: an analysis pipeline for sequence-specific ligation-mediated amplification reads

“…Ligation-Mediated Amplification (LMA) library preparation protocols [1,2] have become increasingly useful in targeted sequencing methodologies. Applications include Carbon Copy-Chromatin Immunoprecipitation (2C-ChIP) [3], used to study protein-DeoxyriboNucleic Acid (DNA) interactions at a defined set of loci, and Chromosome Conformation Capture Carbon Copy (5C) [4], for the targeted analysis of chromatin architecture.…”