The document provides an overview of recombinant DNA technology and cloning techniques. It discusses: 1) The general steps to clone DNA - isolating DNA from an organism, cutting it with restriction enzymes to create recombinant DNA, and introducing the DNA into a host. 2) Types of cloning vectors like plasmids, artificial chromosomes, and viruses that are used to clone DNA fragments. Genomic and cDNA libraries containing clones of all DNA sequences are also described. 3) Techniques for identifying recombinant clones like hybridization probes, complementation of mutations, restriction mapping, and sequencing.

1. Chapter 8: Recombinant DNA Technology Linnea Fletcher Ph.D. BIOL 2316

3. DNA fragments separated by gel electrophoresis and visualized under UV light. (EtBr intercalates between the bases; allowing for visualization of the DNA)

5. Figure 8.1 Restriction site in DNA, showing symmetry of the sequence around the center point. The sequence is a palindrome, reading the same from left to right (5’-to-3’) on the top strand (GAATTC, here) as it does from right to left (5’-to-3’) on the bottom strand. Shown is the restriction site for Eco RI.

6. How are restriction enzymes named?

7. Figure 8.2 Examples of how restriction enzymes cleave DNA. (a) Sma I results in blunt ends. (b) Bam HI results in 5’ overhanging (“sticky”) ends. (c) Pst I results in 3’ overhanging (“sticky”) ends.

8. Figure 8.3 Cleavage of DNA by the restriction enzyme Eco RI. Eco RI makes staggered, symmetrical cuts in DNA, leaving “sticky” ends. A DNA fragment with a sticky end produced by Eco RI digestion can bind by complementary base pairing (anneal) to any other DNA fragment with a sticky end produced by Eco RI cleavage. The gaps can then be sealed by DNA ligase.

10. Must have: 1) Ori 2) A dominant selectable Marker 3) Cleavage sites for cloning 4) (high copy no.) Figure 8.4 The plasmid cloning vector pUC19. This plasmid has an origin of replication ( ori ), an amp R selectable marker, and a polylinker located within part of the  -galactosidase gene lacZ +.

11. Figure 8.5 Insertion of a piece of DNA into the plasmid cloning vector pUC19 to produce a recombinant DNA molecule. The vector pUC19 contains several unique restriction enzyme sites localized in a polylinker that are convenient for constructing recombinant DNA molecules. The insertion of a DNA fragment into the polylinker disrupts part of the  -galactosidase ( lacZ +) gene, leading to nonfunctional  -galactosidase in E. coli . The blue–white color selection test described in the text can be used to select for vectors with or without inserts.

12. Yeast Artificial Chromosome Example of a yeast artificial chromosome (YAC) cloning vector. A YAC vector contains a yeast telomere ( TEL ) at each end, a yeast centromere sequence ( CEN ), a yeast selectable marker for each arm (here, TRP1 and URA3 ), a sequence that allows autonomous replication in yeast ( ARS ), and restriction sites for cloning.

15. Figure 8.7 Use of partial digestion with a restriction enzyme to produce DNA fragments of appropriate size for constructing a genomic library.

16. cDNA Libraries are best for eukaryotes Figure 8.8 The synthesis of double-stranded complementary DNA (cDNA) from a polyadenylated mRNA, using reverse transcriptase, RNase H, DNA polymerase I, and DNA ligase.

17. Cloning of cDNA using BamHI linkers

19. Screening for specific cDNA plasmids in a cDNA Library by using an antibody probe The antibody binds to a specific Site on a protein that is made via The inserted foreign DNA. This Is a Western Blot since it uses antibody To detect a protein.

22. Radioactive Labeling of DNA

23. PowerPoint ® Layered Art Using DNA probes to screen plasmid genomic libraries for specific DNA sequences

24. PowerPoint ® Layered Art Figure 8.11 Using DNA probes to screen plasmid genomic libraries for specific DNA sequences.

25. PowerPoint ® Layered Art Figure 8.11 Using DNA probes to screen plasmid genomic libraries for specific DNA sequences.

26. PowerPoint ® Layered Art

27. Describe how complementation can be used to screen an expression library. What are some limits to the use of this approach for isolating a specific gene? Figure 8.12 Example of cloning a gene by complementation of mutations: cloning of the yeast ARG1 gene.

29. Figure 8.14 An agarose gel electrophoresis apparatus (right) and power supply (left).

31. Figure 8.13 Constructing a restriction map for Eco RI and Bam HI in a DNA fragment.

33. Figure 8.15 Example of restriction mapping to confirm that a plasmid has been constructed correctly. The Eco RI– Eco RI fragment can insert into the pUC19 vector in two alternative orientations. By cutting with Aat II, the site for which is located asymmetrically in the cloned fragment, the orientation of the clones can be distinguished by the restriction fragment sizes.

34. Figure 8.16 Southern blot procedure for analyzing cellular DNA for the presence of sequences complementary to a labeled probe, such as a cDNA molecule made from an isolated mRNA molecule. The hybrids, shown as three bands in this theoretical example, are visualized by autoradiography or chemiluminescence.

37. Figure 8.17 Dideoxy DNA sequencing of a theoretical DNA fragment. (The “number of nucleotides” in the gel analysis refers to nucleotides added to the primer during new DNA synthesis.)

39. Can you read this sequence? TRY IT A 5’ Read from the bottom up Figure 8.19 Autoradiogram of a dideoxy sequencing gel. The letters over the lanes (A, C, G, and T) correspond to the particular dideoxy nucleotide used in the sequencing reaction analyzed in a given lane.

40. Figure 8.20 Results of automated DNA sequence analysis using fluorescent dyes. The procedure is described in the text. The automated sequencer generates the curves shown in the figure from the fluorescing bands on the gel. The colors are generated by the machine and indicate the four bases: A is green, G is black, C is blue, and T is red. Where bands cannot be distinguished clearly, an N is listed.

43. Figure 8.21 The polymerase chain reaction (PCR) for selective amplification of DNA sequences.