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 Bacterial chromosome Plasmid Cell containing gene of interest Gene inserted into plasmid Recombinant DNA (plasmid) Plasmid put into bacterial cell Gene of interest DNA of chromosome Recombinate bacterium Host cell grown in culture, to form a clone of cells containing the “cloned” gene of interest Protein harvested Basic research on protein Basic research and various applications Copies of gene Basic 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 Human growth hormone treats stunted growth Protein expressed by gene of interest 1 2 3 4

Chapter 20: DNA Technology and Genomics How is a gene cut out of a chromosome? Restriction enzymes Recognize a palindrome sequence Originally found in bacteria Overhangs are “sticky ends” & will bind to any complementary sequence DNA ligase makes a recombinant DNA molecule Restriction site DNA 5 3 G A A T T C C T T A A G Restriction enzyme cuts the sugar-phosphate backbones at each arrow DNA fragment from another source is added. Base pairing of sticky ends produces various combinations. DNA ligase seals the strands. Sticky end Fragment from different DNA molecule cut by the same restriction enzyme One possible combination Recombinant DNA molecule G C T T A A A A T T C A A T T C T T A A 1 2 3

Chapter 20: DNA Technology and Genomics How is a gene cut out of a chromosome? How is recombinant DNA cloned? Recombinant DNA plasmids Sticky ends Human DNA Fragments Human cell Gene of interest Bacterial cell ampR gene (ampicillin resistance) Bacterial plasmid Restriction site lacZ gene (lactose breakdown) 1 Isolate plasmid DNA and human DNA. 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.

Chapter 20: DNA Technology and Genomics How is a gene cut out of a chromosome? How is recombinant DNA cloned? Recombinant bacteria Recombinant DNA plasmids Sticky ends Human DNA Fragments Human cell Gene of interest Bacterial cell ampR gene (ampicillin resistance) Bacterial plasmid Restriction site lacZ gene (lactose breakdown) 1 Isolate plasmid DNA and human DNA. 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.

Chapter 20: DNA Technology and Genomics How is a gene cut out of a chromosome? How is recombinant DNA cloned? Colony carrying non- recombinant plasmid with intact lacZ gene Bacterial clone Colony carrying re- combinant plasmid with disrupted lacZ gene Recombinant bacteria Recombinant DNA plasmids Sticky ends Human DNA Fragments Human cell Gene of interest Bacterial cell ampR gene (ampicillin resistance) Bacterial plasmid Restriction site lacZ gene (lactose breakdown) 1 Isolate plasmid DNA and human DNA. 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.

Chapter 20: DNA Technology and Genomics How is a gene cut out of a chromosome? How is recombinant DNA cloned? 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 Recombinant plasmids Recombinant phage DNA Phage clones (b) Phage library (a) Plasmid library or Bacterial clones

Chapter 20: DNA Technology and Genomics 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 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 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) Denature DNA – 95°C Annealing – 65°C Extension – 72°C Repeat this cycle 25 – 35 times Each cycle doubles the DNA

Chapter 20: DNA Technology and Genomics 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 DNA loaded into wells Electrical current applied (-) DNA moves toward (+) Shorter molecules move faster DNA is visualized

Chapter 20: DNA Technology and Genomics 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 Sickle-cell mutant -globin allele 175 bp 201 bp Large fragment DdeI Ddel 376 bp DdeI restriction sites in normal and sickle-cell alleles of -globin gene. Electrophoresis of restriction fragments from normal and sickle-cell alleles. Normal allele Sickle-cell allele 201 bp 175 bp (a) (b) Recognition Site                  

Chapter 20: DNA Technology and Genomics 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 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? What is a northern blot & a western blot northern – detects RNA with nucleic acid probe western – detects protein with an antibody 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 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? What is a northern blot & a western blot How is DNA sequenced? 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 How can gene function be determined? in vitro mutagenesis – disable gene & observe consequences RNA interference (RNAi) – silencing of gene expression by using DS-RNA with matching sequence which triggers breakdown of mRNA.

Chapter 20: DNA Technology and Genomics 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? What is a northern blot & a western blot How is DNA sequenced? What are genomics? How can gene function be determined? in vitro mutagenesis – disable gene & observe consequences RNA interference (RNAi) – silencing of gene expression by using DS-RNA with matching sequence which triggers breakdown of mRNA. 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