Restriction Cutting Independent Method for Cloning Genomic DNA Segments Outside the Boundaries of Known Sequences

Abstract

We present a simple method for cloning genomic DNA segments outside the boundaries of known sequences, which is not dependent on restriction cutting or mapping. In the first step of the method, a library of single-stranded flanking sequences is generated by linear amplification with one primer in the known region. A homooligomeric cytosine tail is added to each of the single-stranded fragments by a terminal transferase catalyzed reaction. The tailed fragments are then amplified by PCR with a nested primer in the known region and a poly-guanine primer complementary to the cytosine tail in the unknown region. Finally, the different fragments are separated by cloning and characterized by sequencing. The method was used to clone both the upstream (5′) and the downstream (3′) genomic regions of an intron-interrupted tRNALeu(UAA) gene from three cyanobacteria belonging to the genus Microcystis.

Comparison of Cyanopeptolin Genes in Planktothrix , Microcystis , and Anabaena Strains: Evidence for Independent Evolution within Each Genus

“…The fragments were cloned by utilizing a TA TOPO cloning kit (Invitrogen), and several overlapping fragments were sequenced by primer walking with an ABI 3730 sequencer and BigDye v3.1 solution. Amplification of unknown flanking regions by linear PCR were followed by oligo(dC) tailing with terminal transferase and PCR with oligo(dG) and a specific primer (35), used to amplify additional NRPS regions (see Table S1 in the supplemental material). Fragments were assembled, and open reading frames (ORFs) were translated using Vector NTI (Invitrogen).…”

Identification of a Putative Operon Involved in Fructooligosaccharide Utilization by Lactobacillus paracasei

“…The ligation products were transformed into E. coli DH5␣, two independent positive clones containing each cloned PCR product were selected, and inserts were sequenced. Regions flanking the contigs of interest were sequenced by chromosome walking using the tailed-PCR technique as described previously by Rudi et al (44). Briefly, a single gene-specific primer was used to amplify the region beyond the known sequence with 25 amplification cycles using the Easy-A High-Fidelity PCR cloning enzyme (Stratagene Corp., La Jolla, CA) in a standard 50-l PCR mixture, which generated a population of single-stranded flanking sequences.…”

Spontaneous mutations in gas vesicle genes of Planktothrix spp. affect gas vesicle production and critical pressure

“…Downstream sequences were obtained using the method of Rudi et al [15]. A library of single-stranded (ss) DNA fragments covering the downstream region was generated by linear ampli¢cation (for 35 cycles) with the gvpC-speci¢c forward primer GVPC9 (annealing temperature 65 ‡C).…”