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Concept 20.1: DNA cloning yields multiple copies of a gene or other DNA segment • To work directly with specific genes, scientists prepare well-defined segments of DNA in identical copies, a process called DNA cloning © 2011 Pearson Education, Inc. DNA Cloning and Its Applications: A Preview • Most methods for cloning pieces of DNA in the laboratory share general features, such as the use of bacteria and their plasmids • Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome • Cloned genes are useful for making copies of a particular gene and producing a protein product © 2011 Pearson Education, Inc. • Gene cloning involves using bacteria to make multiple copies of a gene • Foreign DNA is inserted into a plasmid, and the recombinant plasmid is inserted into a bacterial cell • Reproduction in the bacterial cell results in cloning of the plasmid including the foreign DNA • This results in the production of multiple copies of a single gene © 2011 Pearson Education, Inc. Figure 20.2 Bacterium 1 Gene inserted into plasmid Bacterial Plasmid chromosome Recombinant DNA (plasmid) Cell containing gene of interest Gene of interest 2 Plasmid put into bacterial cell DNA of chromosome (“foreign” DNA) Recombinant bacterium 3 Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Protein expressed from gene of interest Gene of interest Protein harvested Copies of gene Basic research on gene 4 Basic research and various applications Basic research on protein Gene for pest Gene used to alter Protein dissolves Human growth resistance inserted bacteria for cleaning blood clots in heart hormone treats into plants up toxic waste attack therapy stunted growth Figure 20.2a Bacterium 1 Gene inserted into plasmid Bacterial Plasmid chromosome Recombinant DNA (plasmid) Recombinant bacterium Gene of interest 2 Plasmid put into bacterial cell Cell containing gene of interest DNA of chromosome (“foreign” DNA) Figure 20.2b 3 Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Protein expressed from gene of interest Gene of interest Protein harvested Copies of gene Basic research on gene 4 Basic research and various applications Basic research on protein Gene for pest Gene used to alter Protein dissolves Human growth resistance inserted bacteria for cleaning blood clots in heart hormone treats into plants up toxic waste attack therapy stunted growth Using Restriction Enzymes to Make Recombinant DNA • Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites • A restriction enzyme usually makes many cuts, yielding restriction fragments • The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends.” Animation: Restriction Enzymes © 2011 Pearson Education, Inc. • Sticky ends can bond with complementary sticky ends of other fragments • DNA ligase is an enzyme that seals the bonds between restriction fragments © 2011 Pearson Education, Inc. Figure 20.3-3 Restriction site 5 3 GAATTC CTTAAG DNA 5 3 1 Restriction enzyme cuts sugar-phosphate backbones. 5 3 5 3 5 Sticky 3 3 5 end 5 2 DNA fragment added 3 3 5 from another molecule cut by same enzyme. Base pairing occurs. 5 3 5 3 3 DNA ligase 3 5 G AATT C C TTAA G G AATT C C TTAA G 53 5 3 3 5 One possible combination seals strands 5 3 3 Recombinant DNA molecule 5 Cloning a Eukaryotic Gene in a Bacterial Plasmid • In gene cloning, the original plasmid is called a cloning vector • A cloning vector is a DNA molecule that can carry foreign DNA into a host cell and replicate there © 2011 Pearson Education, Inc. Figure 20.4 TECHNIQUE Bacterial plasmid R amp gene Hummingbird cell lacZ gene Restriction site Sticky ends Gene of interest Hummingbird DNA fragments Recombinant plasmids Nonrecombinant plasmid Bacteria carrying plasmids RESULTS Colony carrying nonrecombinant plasmid with intact lacZ gene Colony carrying recombinant plasmid with disrupted lacZ gene One of many bacterial clones Figure 20.4b Bacteria carrying plasmids RESULTS Colony carrying nonrecombinant plasmid with intact lacZ gene Colony carrying recombinant plasmid with disrupted lacZ gene One of many bacterial clones 12.10 Gel electrophoresis sorts DNA molecules by size • Restriction fragments of DNA can be sorted by size Mixture of DNA molecules of different sizes Longer molecules Power source Gel Shorter molecules Glass plates Completed gel Figure 12.10 Screening a Library for Clones Carrying a Gene of Interest • A clone carrying the gene of interest can be identified with a nucleic acid probe having a sequence complementary to the gene • This process is called nucleic acid hybridization © 2011 Pearson Education, Inc. • A probe can be synthesized that is complementary to the gene of interest • For example, if the desired gene is 5 CTCATCACCGGC 3 – Then we would synthesize this probe 3 GAGTAGTGGCCG © 2011 Pearson Education, Inc. 5 • The DNA probe can be used to screen a large number of clones simultaneously for the gene of interest • Once identified, the clone carrying the gene of interest can be cultured © 2011 Pearson Education, Inc. Problems with expressing Eukaryotic genes in Bacteria • Eventhough bacteria are capable of transcribing and translating eukaryotic genes, they do not have the capacity to splice exons and get rid of introns. So in order for them to be able to make a protein, an in intronless gene has to be inserted into bacteria Figure 20.6-5 DNA in nucleus mRNAs in cytoplasm Reverse transcriptase Poly-A tail mRNA A A A A A A 3 5 3 T T T T T 5 DNA Primer strand A A A A A A 3 T T T T T 5 5 3 5 3 DNA polymerase 3 5 3 5 5 3 cDNA 12.11 Restriction fragment analysis is a powerful method that detects differences in DNA sequences • Scientists can compare DNA sequences of different individuals based on the size of the fragments Allele 1 Allele 2 w Cut z x Cut y Figure 12.11A Cut y DNA from chromosomes 1 2 Longer fragments Shorter fragments Figure 12.11B OTHER APPLICATIONS OF DNA TECHNOLOGY 12.15 Connection: DNA technology is used in courts of law • DNA fingerprinting can help solve crimes Defendant’s blood Blood from defendant’s clothes Victim’s blood Figure 12.15A, B 12.12 The PCR method is used to amplify DNA sequences • The polymerase chain reaction (PCR) can quickly clone a small sample of DNA in a test tube Initial DNA segment 1 2 4 Number of DNA molecules 8 Figure 12.12 12.16 Connection: Recombinant cells and organisms can mass-produce gene products • Recombinant cells and organisms are used to manufacture useful proteins Table 12.16