©2011 Elsevier, Inc. Molecular Tools and Infectious Disease Epidemiology Betsy Foxman Chapter 5 A Primer of Molecular Biology

©2011 Elsevier, Inc. Transcription and translation in eukaryotic and prokaryotic cells. Prokaryotic DNA has little or no intragenetic regions (introns), and prokaryotes do not have a nucleus. Source: Reproduced, with permission, from Alberts et al. (2004). FIGURE 5.1

©2011 Elsevier, Inc. Examples of single nucleotide polymorphisms (SNPs), insertion, deletion, and frameshift mutations. Source: Reproduced from the U.S. National Library of Medicine. FIGURE 5.2 Single Nucleotide Polymorphism (SNP) Original DNA code CATCATACATCA Code with a SNP, A is substituted for T CAACATACATCA

©2011 Elsevier, Inc. Map of the positions of different sequence variations found in the M. tuberculosis PE_PGRS16 (Panel A) and PE_PGRS26 (Panel B) genes. The PE domain is shaded in black and the PGRS domain is shaded in light gray. The striped region in Panel (A) represents an additional atypical sequence at the carboxy-terminus of PE_PGRS16. Triangles represent insertions, bold lines represent deletions, open circles represent synonymous SNPs, and solid circles represent nonsynonymous SNPs. The asterisk in Panel (A) labeled FS2 represents a 1-bp deletion. Source: Reproduced, with permission, from Talarico et al. (2008). FIGURE 5.3

©2011 Elsevier, Inc. Meiosis, a type of nuclear division, occurs only in reproductive cells and results in a diploid cell (one having two sets of chromosomes) giving rise to four haploid cells (having a single set of chromosomes). Each haploid cell can subsequently fuse with a gamete of the opposite sex during sexual reproduction. In this illustration, two pairs of homologous chromosomes enter Meiosis I, which results initially in two daughter nuclei, each with two copies of each chromosome. These two cells then enter Meiosis II, producing four daughter nuclei, each with a single copy of each chromosome. Source: Reproduced from A Basic Introduction to the Science Underlying NCBI Resources. FIGURE 5.4

©2011 Elsevier, Inc. (1)Transformation: uptake of free DNA into the cell; (2) Transduction: phage introduces DNA into the cell. (3) \ Conjugation: transfer of plasmids between cells. Source: Reproduced, with permission, from Alekshun and Levy (2007). FIGURE 5.5

©2011 Elsevier, Inc. Electrophoresis. S ource: Reproduced, with permission, from Huntingtons Outreach Project for Education at Stanford. FIGURE 5.6

©2011 Elsevier, Inc. Polymerase chain reaction (PCR). Source: Reproduced, with permission, from Veirstraete (1999). FIGURE 5.7

©2011 Elsevier, Inc. Results from qPCR using a universal bacterial primer on bacterial DNA extracted from human milk specimens. A, Graph shows serially diluted control and unknowns (line beginning at 30 cycles). Line at ~0 on the y-axis is no DNA control; line at ~800 on the y-axis is the threshold cycle. B, Graph shows log of starting DNA quantity by threshold cycle for serially diluted control (open circles) and unknowns (crosses). Source: Authors data. FIGURE 5.8

©2011 Elsevier, Inc. Melting curve from qPCR using a universal bacterial primer on bacterial DNA extracted from human milk specimens. Source: Authors data. FIGURE 5.9

©2011 Elsevier, Inc. Schematic representation of the progress of the enzyme reaction in liquid-phase pyrosequencing. Light is emitted proportional to the number of nucleotides incorporated. Nucleotides are introduced one at a time; a graph is created that shows the intensity of light and the nucleotide introduced. Enzymes are used to follow the incorporation of nucleotides. Source: Reproduced, with permission, from Ronaghi (2001). FIGURE 5.10

©2011 Elsevier, Inc. The basic Sanger method is shown here. In high throughput sequencers, the DNA fragments are made single-stranded then anchored. Fluorescently labeled nucleotides are added, detected, and identified, then the fluorescence is removed. Source: Reproduced, with permission, from Shaw. FIGURE 5.11

©2011 Elsevier, Inc. Example of adding unique DNA identifier (barcode) during PCR amplification before sequencing using the 454 (pyrosequencing) system. This step also adds the primer sequence for the 454 PCR. Source: Reproduced, with permission, from Hoffman et al. (2007). FIGURE 5.12

©2011 Elsevier, Inc. Example of adding unique DNA identifier (barcode) during PCR amplification before sequencing using the 454 (pyrosequencing) system. This step also adds the primer sequence for the 454 PCR. Source: Reproduced, with permission, from Hoffman et al. (2007). FIGURE 5.13