1. Lecture 3 DNA Technologies
Biochemistry
Lecture 3
DNA Technologies

2. FIGURE 9-33 Cloning in vertebrates
FIGURE 9-33 Cloning in vertebrates. Genes for several variants of green fluorescent protein have been introduced into different strains of zebrafish, making each of them literally glow in the dark. Each variant GFP fluoresces in a different part of the light spectrum, making the fish expressing it glow in a particular color (red, green, or yellow).

3. DNA Cloning Introduction into host cell
FIGURE 9-1 Schematic illustration of DNA cloning. A cloning vector and eukaryotic chromosomes are separately cleaved with the same restriction endonuclease. The fragments to be cloned are then ligated to the cloning vector. The resulting recombinant DNA (only one recombinant vector is shown here) is introduced into a host cell where it can be propagated (cloned). Note that this drawing is not to scale: the size of the E. coli chromosome relative to that of a typical cloning vector (such as a plasmid) is much greater than depicted here.

4. FIGURE 9-1 (part 1) Schematic illustration of DNA cloning
FIGURE 9-1 (part 1) Schematic illustration of DNA cloning. A cloning vector and eukaryotic chromosomes are separately cleaved with the same restriction endonuclease. The fragments to be cloned are then ligated to the cloning vector. The resulting recombinant DNA (only one recombinant vector is shown here) is introduced into a host cell where it can be propagated (cloned). Note that this drawing is not to scale: the size of the E. coli chromosome relative to that of a typical cloning vector (such as a plasmid) is much greater than depicted here.

5. FIGURE 9-1 (part 2) Schematic illustration of DNA cloning
FIGURE 9-1 (part 2) Schematic illustration of DNA cloning. A cloning vector and eukaryotic chromosomes are separately cleaved with the same restriction endonuclease. The fragments to be cloned are then ligated to the cloning vector. The resulting recombinant DNA (only one recombinant vector is shown here) is introduced into a host cell where it can be propagated (cloned). Note that this drawing is not to scale: the size of the E. coli chromosome relative to that of a typical cloning vector (such as a plasmid) is much greater than depicted here.

6. Restriction Enzymes
FIGURE 9-2ab Cleavage of DNA molecules by restriction endonucleases. Restriction endonucleases recognize and cleave only specific sequences, leaving either (a) sticky ends (with protruding single strands) or (b) blunt ends. Fragments can be ligated to other DNAs, such as the cleaved cloning vector (a plasmid) shown here. This reaction is facilitated by the annealing of complementary sticky ends. Ligation is less efficient for DNA fragments with blunt ends than for those with complementary sticky ends, and DNA fragments with different (noncomplementary) sticky ends generally are not ligated.

7. FIGURE 9-3 The constructed E. coli plasmid pBR322
FIGURE 9-3 The constructed E. coli plasmid pBR322. Note the location of some important restriction sites—for PstI, EcoRI, BamHI, SalI, and PvuII; ampicillin- and tetracycline-resistance genes; and the replication origin (ori). Constructed in 1977, this was one of the early plasmids designed expressly for cloning in E. coli.

8. FIGURE 9-1 (part 1) Schematic illustration of DNA cloning
FIGURE 9-1 (part 1) Schematic illustration of DNA cloning. A cloning vector and eukaryotic chromosomes are separately cleaved with the same restriction endonuclease. The fragments to be cloned are then ligated to the cloning vector. The resulting recombinant DNA (only one recombinant vector is shown here) is introduced into a host cell where it can be propagated (cloned). Note that this drawing is not to scale: the size of the E. coli chromosome relative to that of a typical cloning vector (such as a plasmid) is much greater than depicted here.

9. PCR Polymerase Chain Reaction
FIGURE 9-16a (part 1) Amplification of a DNA segment by the polymerase chain reaction. (a) The PCR procedure has three steps. DNA strands are 1 separated by heating, then 2 annealed to an excess of short synthetic DNA primers (blue) that flank the region to be amplified; 3 new DNA is synthesized by polymerization. The three steps are repeated for 25 or 30 cycles. The thermostable DNA polymerase TaqI (from Thermus aquaticus, a bacterial species that grows in hot springs) is not denatured by the heating steps.

11. Antibiotic Selection
Antibiotics, such as penicillin and ampicillin, kill bacteria
Plasmids can carry genes that give host bacterium a resistance against antibiotics
Allows growth (selection) of bacteria that have taken up the plasmid

12. Expression of Cloned Genes
FIGURE 9-10 DNA sequences in a typical E. coli expression vector. The gene to be expressed is inserted into one of the restriction sites in the polylinker, near the promoter (P), with the end encoding the amino terminus proximal to the promoter. The promoter allows efficient transcription of the inserted gene, and the transcription termination sequence sometimes improves the amount and stability of the mRNA produced. The operator (O) permits regulation by means of a repressor that binds to it (Chapter 28). The ribosome binding site provides sequence signals needed for efficient translation of the mRNA derived from the gene. The selectable marker allows the selection of cells containing the recombinant DNA.

13. DNA Electrophoresis

14. Sanger DNA Sequencing
FIGURE 8-33b DNA sequencing by the Sanger method. This method makes use of the mechanism of DNA synthesis by DNA polymerases (Chapter 25). (b) The Sanger sequencing procedure uses dideoxynucleoside triphosphate (ddNTP) analogs to interrupt DNA synthesis. (The Sanger method is also known as the dideoxy method.) When a ddNTP is inserted in place of a dNTP, strand elongation is halted after the analog is added, because it lacks the 3′-hydroxyl group needed for the next step.
FIGURE 8-33a DNA sequencing by the Sanger method. This method makes use of the mechanism of DNA synthesis by DNA polymerases (Chapter 25). (a) DNA polymerases require both a primer (a short oligonucleotide strand), to which nucleotides are added, and a template strand to guide selection of each new nucleotide. In cells, the 3′-hydroxyl group of the primer reacts with an incoming deoxynucleoside triphosphate (dNTP) to form a new phosphodiester bond.
FIGURE 8-33c DNA sequencing by the Sanger method. This method makes use of the mechanism of DNA synthesis by DNA polymerases (Chapter 25). (c) The DNA to be sequenced is used as the template strand, and a short primer, radioactively or fluorescently labeled, is annealed to it. By addition of small amounts of a single ddNTP, for example ddCTP, to an otherwise normal reaction system, the synthesized strands will be prematurely terminated at some locations where dC normally occurs. Given the excess of dCTP over ddCTP, the chance that the analog will be incorporated whenever a dC is to be added is small. However, ddCTP is present in sufficient amounts to ensure that each new strand has a high probability of acquiring at least one ddC at some point during synthesis. The result is a solution containing a mixture of labeled fragments, each ending with a C residue. Each C residue in the sequence generates a set of fragments of a particular length, such that the different-sized fragments, separated by electrophoresis, reveal the location of C residues. This procedure is repeated separately for each of the four ddNTPs, and the sequence can be read directly from an autoradiogram of the gel. Because shorter DNA fragments migrate faster, the fragments near the bottom of the gel represent the nucleotide positions closest to the primer (the 5′ end), and the sequence is read (in the 5′→3′ direction) from bottom to top. Note that the sequence obtained is that of the strand complementary to the strand being analyzed.

15. DNA Sequencing

16. Automated DNA Sequencing
FIGURE 8-34 Strategy for automating DNA sequencing reactions. Each dideoxynucleotide used in the Sanger method can be linked to a fluorescent molecule that gives all the fragments terminating in that nucleotide a particular color. All four labeled ddNTPs are added to a single tube. The resulting colored DNA fragments are then separated by size in a single electrophoretic gel contained in a capillary tube (a refinement of gel electrophoresis that allows for faster separations). All fragments of a given length migrate through the capillary gel in a single peak, and the color associated with each peak is detected using a laser beam. The DNA sequence is read by determining the sequence of colors in the peaks as they pass the detector. This information is fed directly to a computer, which determines the sequence.

17. Shotgun Sequencing
FIGURE 9-17 The Human Genome Project strategy. Clones isolated from a genomic library were ordered into a detailed physical map, then individual clones were sequenced by shotgun sequencing protocols. The strategy used by the commercial sequencing effort eliminated the step of creating the physical map and sequenced the entire genome by shotgun cloning.

18. Applications of Sequencing
Solving crimes
Identifying genetic diseases
Huntington’s disease
Breast cancer
Sickle cell anemia
Viral infections
Human migration patterns
Parentage/pedigree/genealogy

19. Genome Editing: CRISPR-Cas9 System
Doudna, JA, Charpentier E. Science. 2014; 346(6213).

20. Electrochemical Sequencing

21. FIGURE 9-18 Genomic sequencing timeline
FIGURE 9-18 Genomic sequencing timeline. Discussions in the mid-1980s led to initiation of the Human Genome Project in Preparatory work, including extensive mapping to provide genome landmarks, occupied much of the 1990s. Separate projects were launched to sequence the genomes of other organisms important to research. The sequencing efforts completed to date include many bacterial species (such as Haemophilus influenzae), yeast (S. cerevisiae), nematode worms (e.g., C. elegans), insects (D. melanogaster and Apis mellifera), plants (A. thaliana and Oryza sativa L.), rodents (Mus musculus and Rattus norvegicus), primates (Homo sapiens and Pan troglodytes), and some nasty human pathogens (e.g., Trichomonas vaginalis). Each genome project has a website that serves as a central repository for the latest data.

22. FIGURE 9-19 Snapshot of the human genome
FIGURE 9-19 Snapshot of the human genome. The chart shows the proportions of our genome made up of various types of sequences.