Figure 1
From: Phase variation and microevolution at homopolymeric tracts in Bordetella pertussis
Detection by PCR/LDR of HPT alleles in bvgS. (A) Schematic of BP370 and BP369 genomic DNA templates aligned with LDR oligonucleotides. Purple text denotes the HPT; green star indicates the FAM fluorophore; cyan text shows non-hybridizing bases used for size discrimination of oligos. HPT length is six cytosines (C6) in BP370, and seven cytosines (C7) in BP369. Covalent bonds (diagonal lines) are catalyzed by DNA ligase only if the discriminating and common oligonucleotides are immediately adjacent when hybridized to the template. (B) Raw capillary electrophoresis data for ligation products (green) and molecular weight standards (red) displayed as if an electrophoretic gel image. Source of template genomic DNA is indicated across the top of the panel. BP370* is a culture derived from BP370 that contains a minority population of C7 phase variants. All reactions contained common oligonucleotide; discriminating oligonucleotides are indicated above each lane (C6 and C7 indicate uniplex reactions, C6 + C7 indicates multiplex). Ligation products (arrows) are labeled as follows. C7 and C6 indicate the products formed by ligation of the common oligonucleotide to the C7 and C6 discriminating oligonucleotides, respectively. C6+1 denotes the product formed by ligation of the common oligonucleotide to a synthesis artifact in the C6 discriminating oligonucleotide preparation that has one extra 3' C. Similarly, C7-1 denotes the product formed by ligation of the common oligonucleotide to a synthesis artifact in the C7 discriminating oligonucleotide preparation that has one fewer 3' C. This notation is used throughout the figures of this manuscript. (C) Raw capillary electrophoresis displayed as in panel B. All lanes are multiplex reactions using C6 and C7 discriminating oligos with the genomic DNA template indicated above each lane. Lanes 3–6 show the ratio of PCR amplified bvgS from BP369 to bvgS from BP370. Ligation products are labeled as in panel B. (D) Partial trace of directly sequenced bvgS PCR product from BP370*. Purple arrow indicates the point in the sequence trace after which peak shadowing, indicative of a mixture of HPT alleles, can be observed.