Lecture 18 - DNA Technology PDF

Chapter 19 DNA Technology © 2021 Pearson Education Ltd. Lecture Presentations by Nicole Tunbridge and Kathleen Fitzpatrick Learning Objectives At the end of this lecture, students should be able to: Describe the function of restriction enzymes and their use in DNA technology Outline gene cloning Des...

Chapter 19 DNA Technology © 2021 Pearson Education Ltd. Lecture Presentations by Nicole Tunbridge and Kathleen Fitzpatrick Learning Objectives At the end of this lecture, students should be able to: Describe the function of restriction enzymes and their use in DNA technology Outline gene cloning Describe briefly polymerase chain reaction (PCR) Illustrate the process of gel electrophoresis Elucidate the applications of DNA technology Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Figure 19.1b © 2021 Pearson Education Ltd. Making Multiple Copies of a Gene or Other DNA Segment To work directly with specific genes, scientists prepare well-defined DNA segments in multiple identical copies by a process called DNA cloning Plasmids are small, circular DNA molecules that replicate separately from the bacterial chromosome Researchers can insert DNA into a plasmid to produce a recombinant DNA molecule, which contains DNA from two different sources 1 Plasmids 2 © 2021 Pearson Education Ltd. human Reproduction of a recombinant plasmid in a bacterial cell results in cloning of the plasmid including the foreign DNA This production of multiple copies of a single gene is a type of DNA cloning called gene cloning A plasmid used to clone a foreign gene is called a cloning vector Cloned genes are useful for making copies of a particular gene and producing a protein product © 2021 Pearson Education Ltd. Genetic Engineering/Biotechnology Methods for making recombinant DNA are central to genetic engineering, the direct manipulation of genes for practical purposes DNA technology has revolutionized biotechnology, the manipulation of organisms or their genetic components to make useful products © 2021 Pearson Education Ltd. Gene cloning and its uses Cell containing gene of interest Bacterium 1 Gene inserted into plasmid Bacterial Plasmid chromosome Recombinant DNA (plasmid) Gene of interest DNA of chromosome 2 Plasmid put into bacterial cell Recombinant bacterium 3 Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Gene of Interest Protein expressed by gene of interest Copies of gene Basic Protein harvested 4 Basic research and various applications research on gene Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Basic research on protein Human growth hormone treats 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 Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends” that bond with complementary sticky ends of other fragments DNA ligase is an enzyme that seals the bonds between restriction fragments Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-3-1 Restriction site DNA 1 5¢ 3¢ n 3¢ 5¢ Restriction enzyme cuts sugar-phosphate backbones. Sticky end Fig. 20-3-2 Restriction site DNA 1 5¢ 3¢ 3¢ 5¢ Restriction enzyme cuts sugar-phosphate backbones. Sticky end 2 DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. One possible combination Fig. 20-3-3 Restriction site DNA 1 5¢ 3¢ 3¢ 5¢ Restriction enzyme cuts sugar-phosphate backbones. Sticky end 2 DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. One possible combination 3 DNA ligase seals strands. Recombinant DNA molecule Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR) The polymerase chain reaction, PCR, can produce many copies of a specific target segment of DNA A three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules The key to PCR is an unusual, heat-stable DNA polymerase called Taq polymerase Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-8 5¢ TECHNIQUE 3¢ Target sequence 3¢ Genomic DNA Requirements: dsDNA, heat-resistant DNA pol all four nucleotides (in excess) two 15-20 nucleotide long ssDNA primers 1 Denaturation 5¢ 5¢ 3¢ 3¢ 5¢ o (94-98 C) 2 Annealing Cycle 1 yields 2 molecules (50-65 oC) Primers 3 Extension (70-80 oC) Cycle 2 yields 4 molecules 2n; after 30 cycles: ~1 billion copies Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence a New nucleotides (5’à3’) Fig. 20-8a Requirements: dsDNA, heat-resistant DNA pol all four nucleotides (in excess) two 15-20 nucleotide long ssDNA primers 5¢ TECHNIQUE 3¢ Target sequence Genomic DNA DNA pol from Thermus aquaticus (Taq polymerase). Functional up to 95 0C. 3¢ 5¢ Fig. 20-8b 1 Denaturation 5¢ 3¢ 3¢ 5¢ (94-98 oC) 2 Annealing Cycle 1 yields 2 molecules (50-65 oC) a Primers 3 Extension (70-80 oC) New nucleotides (5’à3’) Fig. 20-8c Cycle 2 yields 4 molecules 2n; after 30 cycles: ~1 billion copies Fig. 20-8d Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence Within a few hours can make a billion copies of a segment even if it originally made up less than 0.001% of total DNA in the sample PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells Gel Electrophoresis One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis This technique uses a gel as a molecular sieve to separate nucleic acids or proteins by size A current is applied that causes charged molecules to move through the gel Molecules are sorted into “bands” by their size Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Figure 19.6 a buffer provide ions to conduct electricity Analyzing Gene Expression The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made mRNA can be detected by nucleic acid hybridization with complementary molecules These complementary molecules, of either DNA or RNA, are nucleic acid probes © 2021 Pearson Education Ltd. In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism Different probes can be labeled with different fluorescent dyes, sometimes with strikingly beautiful results © 2021 Pearson Education Ltd. Figure 19.9 © 2021 Pearson Education Ltd. DNA mRNA Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time Reverse transcriptase is used to synthesize a complementary DNA (cDNA) copy of each mRNA in the sample A second DNA strand, complementary to the first is synthesized by DNA polymerase e © 2021 Pearson Education Ltd. Figure 19.10 © 2021 Pearson Education Ltd. Next, PCR is used to amplify DNA segments of interest from the cDNAs The products are run on a gel to determine which samples expressed the gene of interest The presence, absence and relative expression of mRNA can be compared © 2021 Pearson Education Ltd. A. Gel electrophoresis Different gene expression levels B. Real-Time RT-PCR Different gene expression levels Applications of DNA technology: DNA Sequencing Relatively short DNA fragments can be sequenced by the dideoxy chain termination method F Modified nucleotides called dideoxyribonucleotides (ddNTP) attach to synthesized DNA strands of different lengths Each type of ddNTP is tagged with a distinct fluorescent label that identifies the nucleotide at the end of each DNA fragment The DNA sequence can be read from the resulting spectrogram Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 20-12 TECHNIQUE DNA (template strand) dNTP Primer DNA polymerase DNA (template strand) Deoxyribonucleotides dATP ddATP dCTP ddCTP dTTP ddTTP dGTP ddGTP usually, 100 times lower concentration than deoxynucleotides Labeled strands Shortest Direction of movement of strands Dideoxyribonucleotides (fluorescently tagged) Longest Longest labeled strand Detector Laser RESULTS Shortest labeled strand Last base of longest labeled strand Last base of shortest labeled strand ddNTP: they terminate DNA strand elongation because it lacks 3’OH required for a phosphodiester bond Application: Diagnosis of Diseases Scientists can diagnose many human genetic disorders by using PCR and sequence-specific primers, then sequencing the amplified product to look for the disease-causing mutation Genetic disorders can also be tested for using genetic markers that are linked to the disease-causing allele © 2021 Pearson Education Ltd. Figure 19.15 SNPs (single nucleotide polymorphisms), single nucleotide variants, are among the most useful genetic markers © 2021 Pearson Education Ltd. Forensic Evidence and Genetic Profiles DNA testing can identify individuals with a high degree of certainty in criminal and paternity cases An individual’s unique set of genetic markers, or genetic profile, can be obtained by analysis of tissue or body fluids Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs) STRs are variations in the number of repeats of specific DNA sequences; they are analyzed by PCR and gel electrophoresis © 2021 Pearson Education Ltd. Figure 19.24 Values refer to size of STR © 2021 Pearson Education Ltd. Editing Genes and Genomes The CRISPR-Cas9 system is a powerful new technique for gene editing in living cells and organisms It is an effective way for researchers to knock out a given gene in order to study what the gene does Modifications of the technique allow researchers to repair a gene that has a mutation (e.g., Gene Therapy) © 2021 Pearson Education Ltd. You should now be able to: 1. Describe the natural function of restriction enzymes and explain how they are used in recombinant DNA technology 2. Outline the procedures for cloning a eukaryotic gene in a bacterial plasmid 3. Describe the polymerase chain reaction (PCR) and explain the advantages and limitations of this procedure 4. Explain how gel electrophoresis is used to analyze nucleic acids 5. Describe the application of DNA technology Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings