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Chapter 20: DNA Technology and Genomics - Lots of different techniques - Many used in combination with each other - Uses information from every chapter so far Figure 20.2 Overview of gene cloning Bacterium 1 Gene inserted Cell containing gene of interest into plasmid Bacterial chromosome Gene of interest Plasmid Recombinant DNA (plasmid) 2 Plasmid put into DNA of chromosome bacterial cell Recombinate bacterium 3 Host cell grown in culture, to form a clone of cells containing the “cloned” gene of interest Gene of interest Copies of gene Basic research on gene Gene for pest resistance inserted into plants Protein expressed by gene of interest Protein harvested 4 Basic research and various applications 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 Chapter 20: DNA Technology and Genomics 1. How is a gene cut out of a chromosome? - Restriction enzymes Restriction site - Recognize a palindrome sequence GAATTC - Originally found in bacteria DNA 5 CTTAAG 3 - Overhangs are “sticky ends” & enzyme cuts will bind to any complementary 1 Restriction the sugar-phosphate backbones at each arrow sequence - DNA ligase makes a recombinant G G DNA molecule 3 5 Sticky end 2 DNA fragment from another source is added. Base pairing of sticky ends produces various combinations. G AATTC C TTAA G G G Fragment from different DNA molecule cut by the same restriction enzyme G AATTC CTTAA G One possible combination 3 DNA ligase seals the strands. Recombinant DNA molecule Chapter 20: DNA Technology and Genomics 1. How is a gene cut out of a chromosome? 2. How is recombinant DNA cloned? 1 Isolate plasmid DNA and human DNA. Bacterial cell lacZ gene (lactose breakdown) Human cell Restriction site ampR gene (ampicillin resistance) 2 Cut both DNA samples with the same restriction enzyme, one that makes a single cut within the lacZ gene and many cuts within the human DNA. 3 Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. Bacterial plasmid Gene of interest Sticky ends Human DNA Fragments Recombinant DNA plasmids Chapter 20: DNA Technology and Genomics 1. How is a gene cut out of a chromosome? 2. How is recombinant DNA cloned? 1 Isolate plasmid DNA and human DNA. Bacterial cell lacZ gene (lactose breakdown) Human cell Restriction site ampR gene (ampicillin resistance) Bacterial plasmid Sticky ends 2 Cut both DNA samples with the same restriction enzyme, one that makes a single cut within the lacZ gene and many cuts within the human DNA. 3 Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. 4 Introduce the DNA into bacterial cells that have a mutation in their own lacZ gene. Gene of interest Human DNA Fragments Recombinant DNA plasmids Recombinant bacteria Chapter 20: DNA Technology and Genomics 1. How is a gene cut out of a chromosome? 2. How is recombinant DNA cloned? 1 Isolate plasmid DNA and human DNA. Bacterial cell lacZ gene (lactose breakdown) Human cell Restriction site ampR gene (ampicillin resistance) Bacterial plasmid Sticky ends 2 Cut both DNA samples with the same restriction enzyme, one that makes a single cut within the lacZ gene and many cuts within the human DNA. 3 Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. 4 Introduce the DNA into bacterial cells that have a mutation in their own lacZ gene. 5 Plate the bacteria on agar containing ampicillin and X-gal. Incubate until colonies grow. Gene of interest Human DNA Fragments Recombinant DNA plasmids Recombinant bacteria Colony carrying nonrecombinant plasmid with intact lacZ gene Colony carrying recombinant plasmid with disrupted lacZ gene Bacterial clone Chapter 20: DNA Technology and Genomics 1. How is a gene cut out of a chromosome? 2. How is recombinant DNA cloned? 3. How are genomes of interest kept in a research lab? - Genomic libraries - Collection of clones in either plasmids or phages Foreign genome cut up with restriction enzyme or Recombinant plasmids Bacterial clones (a) Plasmid library Recombinant phage DNA Phage clones (b) Phage library Chapter 20: DNA Technology and Genomics 1. 2. 3. 4. How is a gene cut out of a chromosome? How is recombinant DNA cloned? How are genomes of interest kept in a research lab? How can we find a “gene of interest” in a genomic library? - Screen a genomic library using a radioactive probe - Nucleic acid probe hybridization Figure 20.5 Nucleic acid probe hybridization Chapter 20: DNA Technology and Genomics 1. 2. 3. 4. 5. How is a gene cut out of a chromosome? How is recombinant DNA cloned? How are genomes of interest kept in a research lab? How can we find a “gene of interest” in a genomic library? What is cDNA & how is it made? - complementary DNA - complementary to mRNA - Only exons present - Isolate mRNA - Use reverse transcriptase to make cDNA - cDNA libraries made from different tissues, stages of development, in response drugs, etc Chapter 20: DNA Technology and Genomics 1. 2. 3. 4. 5. 6. How is a gene cut out of a chromosome? How is recombinant DNA cloned? How are genomes of interest kept in a research lab? How can we find a “gene of interest” in a genomic library? What is cDNA & how is it made? What is PCR & how is it used? - Polymerase chain reaction - Used to amplify DNA - Forensics - Paternity testing - To aid in DNA sequencing Figure 20.7 The polymerase chain reaction (PCR) Making DNA - Template - Primers - dNTPs - DNA polymerase (Taq – heat resistant) 1. Denature DNA – 95°C 2. Annealing – 65°C 3. Extension – 72°C Repeat this cycle 25 – 35 times Each cycle doubles the DNA Chapter 20: DNA Technology and Genomics 1. 2. 3. 4. 5. 6. 7. How is a gene cut out of a chromosome? How is recombinant DNA cloned? How are genomes of interest kept in a research lab? How can we find a “gene of interest” in a genomic library? What is cDNA & how is it made? What is PCR & how is it used? What is gel electrophoresis? - Method to separate DNA or protein based on size & charge - Forest analogy…. Figure 20.8 Gel Electrophoresis 1. 2. 3. 4. 5. DNA loaded into wells Electrical current applied (-) DNA moves toward (+) Shorter molecules move faster DNA is visualized Chapter 20: DNA Technology and Genomics 1. 2. 3. 4. 5. 6. 7. 8. How is a gene cut out of a chromosome? How is recombinant DNA cloned? How are genomes of interest kept in a research lab? How can we find a “gene of interest” in a genomic library? What is cDNA & how is it made? What is PCR & how is it used? What is gel electrophoresis? What is RFLP analysis? - Restriction Fragment Length Polymorphism - Combines restriction digest & gel electrophoresis Figure 20.9 Using restriction fragment analysis to distinguish the normal and sickle-cell alleles of the -globin gene Normal -globin allele 201 bp 175 bp DdeI DdeI Recognition Site Large fragment DdeI DdeI Sickle-cell mutant -globin allele Large fragment 376 bp Ddel Ddel Ddel (a) DdeI restriction sites in normal and sickle-cell alleles of -globin gene. Normal Sickle-cell allele allele Large fragment 376 bp 201 bp 175 bp (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles. Chapter 20: DNA Technology and Genomics 1. 2. 3. 4. 5. 6. 7. 8. 9. How is a gene cut out of a chromosome? How is recombinant DNA cloned? How are genomes of interest kept in a research lab? How can we find a “gene of interest” in a genomic library? What is cDNA & how is it made? What is PCR & how is it used? What is gel electrophoresis? What is RFLP analysis? What is Southern blot analysis? - Combination of RFLP & nucleic acid probe hybridization - Transfers DNA from gel to a solid substrate (nitrocellulose paper) Figure 20.10 Southern blotting of DNA fragments Chapter 20: DNA Technology and Genomics 1. How is a gene cut out of a chromosome? 2. How is recombinant DNA cloned? 3. How are genomes of interest kept in a research lab? 4. How can we find a “gene of interest” in a genomic library? 5. What is cDNA & how is it made? 6. What is PCR & how is it used? 7. What is gel electrophoresis? 8. What is RFLP analysis? 9. What is Southern blot analysis? 10. What is a northern blot & a western blot - northern – detects RNA with nucleic acid probe - western – detects protein with an antibody 11. How is DNA sequenced? - Dideoxy termination method - 3’ –OH is missing; therefore, no extension & termination occurs - Combines PCR, electrophoresis, & fluorescent labeling Figure 20.12 Dideoxy chain-termination method for sequencing DNA Reagents needed to make DNA + dideoxy nucleotides What ever color is detected is the last nucleotide. No extension off of dideoxy nucleotide Chapter 20: DNA Technology and Genomics 1. How is a gene cut out of a chromosome? 2. How is recombinant DNA cloned? 3. How are genomes of interest kept in a research lab? 4. How can we find a “gene of interest” in a genomic library? 5. What is cDNA & how is it made? 6. What is PCR & how is it used? 7. What is gel electrophoresis? 8. What is RFLP analysis? 9. What is Southern blot analysis? 10. What is a northern blot & a western blot 11. How is DNA sequenced? 12. What are genomics? - The study of whole sets of genes and their interactions - Human Genome Project has provided sequence – now we must determine how genes interact 13. How can gene function be determined? - in vitro mutagenesis – disable gene & observe consequences - RNA interference (RNAi) – silencing of gene expression by using DSRNA with matching sequence which triggers breakdown of mRNA. Chapter 20: DNA Technology and Genomics 1. How is a gene cut out of a chromosome? 2. How is recombinant DNA cloned? 3. How are genomes of interest kept in a research lab? 4. How can we find a “gene of interest” in a genomic library? 5. What is cDNA & how is it made? 6. What is PCR & how is it used? 7. What is gel electrophoresis? 8. What is RFLP analysis? 9. What is Southern blot analysis? 10. What is a northern blot & a western blot 11. How is DNA sequenced? 12. What are genomics? 13. How can gene function be determined? - in vitro mutagenesis – disable gene & observe consequences - RNA interference (RNAi) – silencing of gene expression by using DSRNA with matching sequence which triggers breakdown of mRNA. 14.What is a DNA microarray? - Method used to measure expression of thousands of genes at once - Uses cDNA to bind to gene segments on a glass slide Figure 20.14 Research Method DNA microarray assay of gene expression levels