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Bacterium

Bacterium. Cell containing gene of interest. Gene inserted into plasmid. 1. 2. 4. 3. Gene of interest. Plasmid. Bacterial chromosome. DNA of chromosome. Recombinant DNA (plasmid). Plasmid put into bacterial cell. Recombinate bacterium.

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Bacterium

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  1. Bacterium Cell containing geneof interest Gene inserted into plasmid 1 2 4 3 Gene of interest Plasmid Bacterialchromosome DNA ofchromosome RecombinantDNA (plasmid) Plasmid put into bacterial cell Recombinatebacterium Host cell grown in culture,to form a clone of cellscontaining the “cloned”gene of interest Gene of interest Protein expressedby gene of interest Copies of gene Protein harvested Basic research and various applications Basic research on protein Basic research on gene Gene used to alterbacteria for cleaningup toxic waste Gene for pestresistance inserted into plants Human growth hormone treatsstunted growth Protein dissolvesblood clots in heartattack therapy Figure 20.2 Overview of gene cloning

  2. Restriction site 3 1 2 5 3 G A A T T C DNA 5 3 C T T A A G Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow G A A T T C C T T A A G Sticky end A A T T C G G DNA fragment from another source is added. Base pairing of sticky ends produces various combinations. C T T A A Fragment from differentDNA molecule cut by thesame restriction enzyme G A A T T C A A T T C G C T T A A T T A A C G G One possible combination DNA ligaseseals the strands. Recombinant DNA molecule 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

  3. Bacterial cell lacZ gene (lactose breakdown) 2 3 1 Human cell Isolate plasmid DNA and human DNA. Restriction site ampR gene (ampicillin resistance) Bacterial plasmid Gene of interest Stickyends Human DNA Fragments 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. Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. Recombinant DNA plasmids Chapter 20: DNA Technology and Genomics • How is a gene cut out of a chromosome? • How is recombinant DNA cloned?

  4. Bacterial cell lacZ gene (lactose breakdown) 3 1 4 2 Human cell Isolate plasmid DNA and human DNA. Restriction site ampR gene (ampicillin resistance) Bacterial plasmid Gene of interest Stickyends Human DNA Fragments 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. Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. Recombinant DNA plasmids Introduce the DNA into bacterial cells that have a mutation in their own lacZ gene. Recombinantbacteria Chapter 20: DNA Technology and Genomics • How is a gene cut out of a chromosome? • How is recombinant DNA cloned?

  5. Bacterial cell lacZ gene (lactose breakdown) 4 1 3 5 2 Human cell Isolate plasmid DNA and human DNA. Restriction site ampR gene (ampicillin resistance) Bacterial plasmid Gene of interest Stickyends Human DNA Fragments 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. Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. Recombinant DNA plasmids Introduce the DNA into bacterial cells that have a mutation in their own lacZ gene. Recombinantbacteria Plate the bacteria on agar containing ampicillin and X-gal. Incubate until colonies grow. Colony carrying re-combinant plasmidwith disrupted lacZ gene Colony carrying non-recombinant plasmid with intact lacZ gene Bacterialclone Chapter 20: DNA Technology and Genomics • How is a gene cut out of a chromosome? • How is recombinant DNA cloned?

  6. Foreign genome cut up with restriction enzyme or Recombinantplasmids Bacterialclones Recombinantphage DNA Phageclones (a) Plasmid library (b) Phage library 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

  7. 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

  8. Figure 20.5 Nucleic acid probe hybridization

  9. 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 processed mRNA • Only exons present • Isolate mRNA • Use reverse transcriptase to make cDNA • cDNA libraries are important…no INTRONS, so can be directly • inserted into bacteria for protein production!

  10. 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

  11. Figure 20.7 The polymerase chain reaction (PCR) • Making DNA • Template • Primers • dNTPs (free nucleotides—A, T, C, G) • DNA polymerase (Taq – heat resistant) • Denature DNA – 95°C • Annealing – allow primers to bind • Extension – polymerase builds new DNA • Repeat this cycle 25 – 35 times • Each cycle doubles the DNA

  12. 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….

  13. Figure 20.8 Gel Electrophoresis • DNA loaded into wells • Electrical current applied • (-) DNA moves toward (+) • Shorter molecules move faster • DNA is visualized

  14. 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

  15. Normal  -globin allele 201 bp Large fragment 175 bp DdeI DdeI DdeI DdeI Sickle-cell mutant -globin allele Large fragment 376 bp Ddel Ddel Ddel (a) DdeIrestriction sites in normal and sickle-cell alleles of -globin gene. Sickle-cellallele Normalallele Largefragment 376 bp 201 bp175 bp (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles. Figure 20.9 Using restriction fragment analysis to distinguish the normal and sickle-cell alleles of the -globin gene

  16. RFLP: Somewhat outdated. What’s next? -RFLP can’t be used for degraded DNA or very small amounts of DNA, so… -STR (short tandem repeats) analysis has replaced RFLP in forensic science. -Look at both parental versions of known STRs in DNA and COUNT how many repeats exist.  Compare suspect with crime scene evidence for match! LOCUS BELOW would be designated (7, 8)

  17. RFLP: Somewhat outdated. What’s next? -The more loci are compared, the more accurate the results.  Comparisons at 13 loci are sufficient to positively ID a suspect. CODIS: Combined DNA Index System

  18. RFLP: Somewhat outdated. What’s next? -Below: Two different STR analyses (4 loci in chart, 3 loci in gel) to determine paternity.

  19. 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)

  20. Figure 20.10 Southern blotting of DNA fragments

  21. 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

  22. 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

  23. 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 miRNA/siRNA with matching sequence which triggers breakdown of mRNA.

  24. 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

  25. Figure 20.14 Research Method DNA microarray assay of gene expression levels

  26. Homework… • Carefully read Section 20.5 (Applications of DNA Technology) on pp. 402-408!! • PCR used to detect HIV and genetic diseases • RFLP analysis used to detect Huntington’s • Gene therapy (“replacing” defective genes) • Making drugs using cloning methods • Insulin, HGH, etc. • Crime scene “DNA fingerprinting” • Utilizes Southern Blotting • “Pharm” animals • Genetically-modified crops

  27. Genetically-modified crops!!! • “Pharm” Animals!!!

  28. Crime Scene DNA (RFLP): O.J. Simpson

  29. Human Genome Project

  30. RFLP analysis of a family w/ genetic disease (sickle cell)

  31. Gene Therapy

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