Recombinant DNA

1. Recombinant DNA Chapter 18

2. Learning Objectives • Define Clone and DNA Cloning • List the three steps of production of recombinant DNA • Describe the characteristics and uses of a restriction endonuclease • Diagram the process of identifying a transformed bacterial colony containing a gene of interest using Ampicillin Resistance, Lactose Metabolism plasmids and nucleic acid hybridization probes

3. Learning Objectives • Explain the uses of RFLPs • Describe the process of producing a transgenic organism, and explain its usefulness

4. DNA cloning • Clone: genetically identical cells or individuals derived from a single ancestor • DNA cloning: a method of producing a large amount of the DNA of interest • Large amounts of identical pieces of DNA enable us to manipulate and recombine genetic material

5. DNA Technologies • DNA technologies are used in molecular testing for many human genetic diseases • DNA fingerprinting used to identify human individuals and individuals of other species • Genetic engineering uses DNA technologies to alter the genes of a cell or organism • DNA technologies and genetic engineering are a subject of public concern

6. Recombinant DNA • DNA from two or more sources joined together • DNA of interest can be spliced into bacterial plasmids (recombination) • Plasmids replicate (amplification) • Plasmids (DNA) are extracted (isolation)

7. Endonucleases • Restriction enzymes(endunucleases) cut DNA at specific sequences in restriction sites • Restriction fragments result • Sticky ends have unpaired bases at cuts which will hydrogen bond • Ligase stitches together paired sticky ends

8. Restriction site for EcoRI DNA EcoRI restriction enzyme cleaves sugar–phosphate backbones at arrows. 1 Sticky end Sticky end 2 DNA fragments with the same sticky ends can pair. Shown here is a DNA fragment inserting between two other DNA fragments, as happens when inserting a DNA fragment into a bacterial plasmid. Another DNA fragment produced by EcoRI digestion Nick in sugar–phosphate backbone Nicks in sugar– phosphate backbones are sealed by DNA ligase. 3 Recombinant DNA molecule Fig. 18-3, p. 374

9. Recombinant DNA • Restriction endonucleases • Each type is specific for a four to eight base pair long palindromic recognition sequence of DNA • Palindrome- reads the same on each strand 3’ to 5’ like GAATTC CTTAAG

10. Gene of interest Restriction site lacZ+ gene ampR gene Cell Plasmid cloning vector DNA fragments with sticky ends Cut plasmid cloning vectors with a restriction enzyme to produce sticky ends Fig. 18-4a, p. 375

11. Inserted genomic DNA fragment Recombinant DNA molecules 4 Introduce recombinant molecules into bacterial cells; each bacterium receives a different plasmid. As the bacteria grow and divide, the recombinant plasmids replicate, thereby amplifying the piece of DNA inserted into the plasmid. Bacterium Bacterial chromosome Progeny bacteria 5 Identify the bacterium containing the plasmid with the gene of interest inserted into it. Grow that bacterium in culture to produce large amounts of the plasmid for experiments with the gene of interest. Fig. 18-2b, p. 373

12. Recombinant DNA • Break cells and use restriction enzyme to isolate DNA of interest (prokaryotic or eukaryotic) • Insert into plasmid (recombination) • Transform into bacteria (replication) • Not very efficient, so for the third step (isolation)- you need to have engineered a way to find the bacteria of interest

13. Four possibilities 1. Desired outcome: plasmid, lac+ broken, gene of interest inserted 2. Bacteria transformed with plasmid, but wrong gene inserted 3. Bacteria transformed with plasmid only- no gene at all inserted 4. Bacteria not transformed

14. Resealed plasmid cloning vector with no inserted DNA fragment Inserted DNA fragment without gene of interest Inserted DNA fragments with gene of interest Recombinant plasmids Nonrecombinant plasmid Fig. 18-4b, p. 375

15. Recombinant DNA • Insert into special screening plasmid- which contains the same restriction enzyme site used above, located in a lacZ gene. • For recombination screening the lacZ gene is broken successfully, it will be white. If not, it will be blue. • The plasmid also contain ampicillin resistance • If transformation worked, the bacteria will grow on plates containing ampicillin. Those who were not transformed will not grow.

16. Bacteria not transformed with a plasmid Bacteria transformed with plasmids Selection: Transformed bacteria grow on medium containing ampicillin because of ampRgene on plasmid. Untransformed bacterium cannot grow on medium containing ampicillin. Screening: Blue colony contains bacteria with a non-recombinant plasmid; that is, the lacZ+ gene is intact. Plate containing ampicillin and X-gal White colony contains bacteria with a recombinant plasmid; that is, the vector with an inserted DNA fragment. Once the white colony with the gene of interest is identified, it can be grown in culture to produce large quantities of the plasmid. Fig. 18-4c, p. 375

17. DNA Hybridization • Uses nucleic acidprobe to identify gene of interest in set of clones • Probe has tag for detection • Identified colony produces large quantities of cloned gene

18. Bacterial colony Culture medium containing ampicillin Filter paper Replica of bacterial colonies Filter paper Fig. 18-5a, p. 377

19. Labeled probe (single stranded) Plasmid DNA (single stranded) Labeled single-stranded DNA probe for the gene of interest Bag Filter Hybridization has occurred between the labeled probe and the plasmids released from the bacteria in this colony. The hybridization is detected in subsequent steps. Fig. 18-5b, p. 377

20. Corresponds to one colony on master plate Developed photographic film Original master plate Fig. 18-5c, p. 377

21. 4 Possibilities

22. How else do we use Restriction Endonuclease?

23. RFLPs • Restriction fragment length polymorphisms • DNA sequence length changes due to varying restriction sites from same region of genome • Sickle cell anemia has RFLPs • Southern blot analysis uses electrophoresis, blot transfer, and labeled probes to identify RFLPs • Alternative is PCR and electrophoresis

24. β-Globin gene 175 bp 201 bp Normal allele MstII MstII MstII 376 bp Sickle-cell mutant allele MstII MstII Region of probe used to screen for sickle-cell mutation Fig. 18-8, p. 381

25. DNA Fingerprinting • Distinguishes between individuals • Uses PCR at multiple loci within genome • Each locus heterozygous or homzygous for short tandem repeats (STR) • PCR amplifies DNA from STR • Number of gel electrophoresis bands shows amplified STR alleles • 13 loci commonly used in human DNA fingerprinting

26. Forensics and Ancestry • Forensics compares DNA fingerprint from sample to suspect or victim • Usually reported as probability DNA came from random individual • Common alleles between children and parents used in paternity tests • Same principle used to determine evolutionary relationships between species

27. a. Alleles at an STR locus STR locus Left PCR primer DNA 9 repeats Right PCR primer 3 different alleles 11 repeats 15 repeats Fig. 18-10a, p. 383

28. b. DNA fingerprint analysis of the STR locus by PCR B C A Cells of three individuals Extract genomic DNA and use specific primers to amplify the STR locus using the PCR. Anyalyze PCR product by gel electrophoresis A B C Positions corresponding to alleles of STR locus 15 11 9 11,11 15,9 11,9 Fig. 18-10b, p. 383

29. Genetic Engineering • Transgenic organisms • Modified to contain genes from external source • Expression vector has promoter in plasmid for production of transgenic proteins in E. coli • Example: Insulin • Protocols to reduce risk of escape

30. Animal Genetic Engineering • Transgenic animals used in research, correcting genetic disorders, and protein production • Germ-line cell transgenes can be passed to offspring (somatic can not) • Embryonic germ-line cells cultured in quantity, made into sperm or eggs • Stem cells

31. Germ-line cells derived from mouse embryo Transgene Cell with transgene Pure population of transgenic cells Fig. 18-11a, p. 385

32. Mice have transgenic cells in body regions including germ line Genetically engineered offspring—all cells transgenic Fig. 18-11b, p. 385

33. Gene Therapy Attempts to correct genetic disorders • Germ-line gene therapy can’t be used on humans • Somatic gene therapy used in humans • Mixed results in humans • Successes for adenosine deaminase deficiency (bubble kid) and sickle-cell • Deaths from immune response and leukemia-like conditions • http://history.nih.gov/exhibits/genetics/sect4.htm

34. Animal Genetic Engineering • “Pharm” animals produce proteins for humans • Usually produced in milk for harmless extraction • Cloned mammals produced by implantation of diploid cell fused with denucleated egg cell • Low cloning success rate • Increased health defects in clones • Gene expression regulation abnormal

35. Learning Objectives • Define Clone and DNA Cloning • List the three steps of production of recombinant DNA • Describe the characteristics and uses of a restriction endonuclease • Diagram the process of identifying a transformed bacterial colony containing a gene of interest using Ampicillin Resistance, Lactose Metabolism plasmids and nucleic acid hybridization probes

36. Learning Objectives • Explain the uses of RFLPs • Describe the process of producing a transgenic organism, and explain its usefulness