Adaptor long-range PCR procedure for clone-specific characterization and chromosomal localization.
An efficient adaptor long-range PCR (ALR-PCR) procedure was developed to detect genomic rearrangements in high-plasticity genomic regions between closely related strains of bacteria. The method was precisely optimized using a combination of high-speed experimental steps for the chromosomal localization and elucidation of deletions, inversions, duplications, or inserted sequences within a clone-specific flanking region. The advantages of this strategy are: (i) ready-to-use polymerase mixtures and Master mix (ready-to-use reaction mixtures with polymerase MasterAmp and buffer 2x Premix 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; (vi) two-step PCR cycling with the same annealing and extension temperature at 68 degrees C; (vii) simple design of the adaptors according to the preferred restriction endonuclease enzyme; and (viii) simple technology and equipment required. The application of this method for a tester-specific suppressive subtractive hybridization (SSH) clone of Brucella melitensis 16M revealed an 837-bp deletion and a 7255-bp DNA transfer from one chromosomal location to another for Brucella abortus 2308 used as a driver.
Tsoktouridis
Georgios
G
Institute of Molecular Biology and Medicine, The University of Scranton, Scranton, PA 18510-4625, USA. gtsok1@yahoo.co.uk
Merz
Christian A
CA
DelVecchio
Vito G
VG
D001483:Base Sequence; D002874:Chromosome Mapping / Q000379:methods*; D003001:Cloning, Molecular; D017931:DNA Primers; D016133:Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers