Fig. 4:

Identification of acquired DNA using BCGH (17). (a) 9 X 10 cm area of the blot hybridized with the H846 probe is shown. The orange box represents the region displayed in ((b), (c), (d)). (b) An image from hybridization with the H103 probe was coded green. (c) An image from the H846 probe was coded red. (d) An overlay of the two displays ((b), (c)) is shown. Arrows in (c) and (d) indicate a spot that is unique to strain H846. (e), Close-up of the spot (red box) that was excised and cloned from a radiolabelled parallel gel run. Ten micrograms of radiolabeled Hinf I digested H846 DNA was resolved using 2DDE and the resulting gel was dried and exposed to film. Using the autoradiogram as a reference, the spot of interest was excised from the dried gel. Re-exposure of the gel post-excision verified that the correct spot was retrieved. The gel spot was hydrated, boiled, crushed and centrifuged. The eluted DNA was precipitated with ethanol in the presence of 10 μg of glycogen as a carrier. Adaptors were ligated to the recovered DNA in a 10 μl reaction volume [containing 20% of the eluted DNA, 200 units T4 DNA ligase (New England Biolabs) and 100 pmoles each of oligonucleotide 5’-CAGGCACCCGGGAGATCTGAATTC-3’ and 5’-ANTGAATTCAGATC-3’] for 12 hours at 16°C, and purified with the Qiaquick PCR purification kit (Qiagen, Mississauga, Ontario). One-fifth volume of the purified DNA was amplified by PCR using the longer oligonucleotide listed above [denaturation at 94° for 4 minutes followed by 35 cycles (94° 1 minute, 52° 1 minute, 72° 1 minute)]. Gel purified PCR product was digested with EcoR I and cloned into pBlueScriptKS+ vector (Stratagene, La Jolla, CA) for sequence analysis. (f), Sequence derived from this spot. Reprinted from Journal of Molecular Biology, Volume 312, Malloff CA, Fernandez RC, Lam WL. Bacterial Comparative Genomic Hybridization: A Method for Directly Identifying Lateral Gene Transfer. Pages 1-5. Copyright (2001) with permission from Elsevier Science.