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GENE CLONING Copyright © 2009 Pearson Education, Inc.

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1 GENE CLONING Copyright © 2009 Pearson Education, Inc.

2 12.1 Genes can be cloned in recombinant plasmids  Genetic engineering involves manipulating genes for practical purposes –Gene cloning leads to the production of multiple identical copies of a gene-carrying piece of DNA –Recombinant DNA is formed by joining DNA sequences from two different sources –One source contains the gene that will be cloned –Another source is a gene carrier, called a vector –Plasmids (small, circular DNA molecules independent of the bacterial chromosome) are often used as vectors Copyright © 2009 Pearson Education, Inc.

3  Steps in cloning a gene 1.Plasmid DNA is isolated 2.DNA containing the gene of interest is isolated 3.Plasmid DNA is treated with restriction enzyme that cuts in one place, opening the circle 4.DNA with the target gene is treated with the same enzyme and many fragments are produced 5.Plasmid and target DNA are mixed and associate with each other Copyright © 2009 Pearson Education, Inc Genes can be cloned in recombinant plasmids

4 6.Recombinant DNA molecules are produced when DNA ligase joins plasmid and target segments together 7.The recombinant DNA is taken up by a bacterial cell 8.The bacterial cell reproduces to form a clone of cells Copyright © 2009 Pearson Education, Inc Genes can be cloned in recombinant plasmids

5 Examples of gene use Recombinant DNA plasmid E. coli bacterium Plasmid Bacterial chromosome Gene of interest DNA Gene of interest Cell with DNA containing gene of interest Recombinant bacterium Clone of cells Genes may be inserted into other organisms Genes or proteins are isolated from the cloned bacterium Harvested proteins may be used directly Examples of protein use Gene of interest Isolate plasmid 1 Isolate DNA 2 Cut plasmid with enzyme 3 Cut cell’s DNA with same enzyme 4 Combine targeted fragment and plasmid DNA 5 Add DNA ligase, which closes the circle with covalent bonds 6 Put plasmid into bacterium by transformation 7 Allow bacterium to reproduce 8 9

6 12.2 Enzymes are used to “cut and paste” DNA  Restriction enzymes cut DNA at specific sequences Restriction enzymes –Each enzyme binds to DNA at a different restriction site –Many restriction enzymes make staggered cuts that produce restriction fragments with single-stranded ends called “sticky ends” –Fragments with complementary sticky ends can associate with each other, forming recombinant DNA  DNA fragments joined together Copyright © 2009 Pearson Education, Inc.

7 Restriction enzyme recognition sequence 1 2 DNA Restriction enzyme cuts the DNA into fragments Sticky end

8 Restriction enzyme recognition sequence 1 2 DNA Restriction enzyme cuts the DNA into fragments Sticky end 3 Addition of a DNA fragment from another source

9 Restriction enzyme recognition sequence 1 2 DNA Restriction enzyme cuts the DNA into fragments Sticky end 3 Addition of a DNA fragment from another source 4 Two (or more) fragments stick together by base-pairing

10 Restriction enzyme recognition sequence 1 2 DNA Restriction enzyme cuts the DNA into fragments Sticky end 3 Addition of a DNA fragment from another source 4 Two (or more) fragments stick together by base-pairing DNA ligase pastes the strands Recombinant DNA molecule 5

11 GENETICALLY MODIFIED ORGANISMS These are organisms that have had foreign genes inserted into their genome through plasmids created by humans Copyright © 2009 Pearson Education, Inc.

12 12.6 Recombinant cells and organisms can mass-produce gene products  Cells and organisms containing cloned genes are used to manufacture large quantities of gene products  Remember how long it took us to create mRNA and the protein hemoglobin  A cell can do this at a rate of 50 nts/sec  Capabilities of the host cell are matched to the characteristics of the desired product Copyright © 2009 Pearson Education, Inc.

13 12.6 Recombinant cells and organisms can mass-produce gene products  Prokaryotic host: E. coli –Can be engineered to secrete proteins  Eukaryotic hosts –Yeast: S. cerevisiae –Can produce and secrete complex eukaryotic proteins –Mammalian cells in culture –Can attach sugars to form glycoproteins –“Pharm” animals –Will secrete gene product in milk Copyright © 2009 Pearson Education, Inc.

14 How will this technology help humans?  What are some products that would be difficult for us to synthesize but easy for a genetically engineered organism to produce? Copyright © 2009 Pearson Education, Inc.

15 How will this technology help humans?  What are some products that would be difficult for us to synthesize but easy for a genetically engineered organism to produce? Copyright © 2009 Pearson Education, Inc. ProductUse InsulinDiabetes Growth hormonesGrowth defects, burns, weight gain in cattle Il-2Cancer TaxolOvarian cancer Hepatitis B vaccine TPAHeart attacks and strokes

16 ProsCons Genetic Modification of Organisms

17 ProsCons Food that can deliver vaccines - bananas that produce hepatitis B vaccine Potential human health impact: allergens, transfer of antibiotic resistance markers, unknown effects More nutritious foods - rice with increased iron and vitamins Potential environmental impact: unintended transfer of transgenes through cross-pollination, unknown effects on other organisms (e.g., soil microbes), and loss of flora and fauna biodiversity Faster growing fish, fruit and nut treesDomination of world food production by a few companies Drought resistantIncreasing dependence on industralized nations by developing countries Resistant to herbicides and pestsViolation of natural organisms’ intrinsic values Mass produce proteins such as insulinTampering with nature by mixing genes among species Clean up the environmentObjections to consuming animal genes in plants and vice versa Stress for animal Labeling not mandatory in some countries (e.g., United States) Genetic Modification of Organisms

18 Real Life CSI Copyright © 2009 Pearson Education, Inc.

19 Not always like you see on TV Copyright © 2009 Pearson Education, Inc. "We asked medical students 'Where did you get some of your ideas before you even came into the medical profession?' And interestingly enough, 'ER came up as the number one influence," Brindley told Canada AM. Knowing that ER was such an influence on the students, Brindley and Needham decided to watch a few episodes of the show and observe their techniques. "We watched two seasons of ER and not once was the resuscitation done properly," Brindley notes. "And that's despite having numerous medical experts advising the show." Real doctors influenced by TV dramas, study suggests March 26,2009

20 How do real detectives find out who did it or who didn’t do it? Copyright © 2009 Pearson Education, Inc.

21 DNA PROFILING Copyright © 2009 Pearson Education, Inc.

22 12.11 The analysis of genetic markers can produce a DNA profile  DNA profiling is the analysis of DNA fragments to determine whether they come from a particular individual –Compares genetic markers from noncoding regions that show variation between individuals –Involves amplification (copying) of markers for analysis –Sizes of amplified fragments are compared Copyright © 2009 Pearson Education, Inc.

23 Crime scene DNA isolated 1 Suspect 1Suspect 2 DNA of selected markers amplified 2 Amplified DNA compared 3

24 12.13 Gel electrophoresis sorts DNA molecules by size  Gel electrophoresis separates DNA molecules based on size –DNA sample is placed at one end of a porous gel –Current is applied and DNA molecules move from the negative electrode toward the positive electrode –Shorter DNA fragments move through the gel pores more quickly and travel farther through the gel –DNA fragments appear as bands, visualized through staining or detecting radioactivity or fluorescence –Each band is a collection of DNA molecules of the same length Copyright © 2009 Pearson Education, Inc. Video: Biotechnology Lab

25 Mixture of DNA fragments of different sizes Completed gel Longer (slower) molecules Gel Power source Shorter (faster) molecules

26 Gilbert Alejandro (Uvalde County, Texas) Factual background. On the evening of April 27, 1990, a woman in her fifties came home and was attacked from behind by a man. The man placed a pillow over her head and sexually assaulted her. He then fled the house. The woman could not describe the man except for basic physical size. She also noted that the man was wearing some kind of cap, a gray T-shirt, and dark-colored shorts. The police canvassed the area and questioned three men, one of whom was wearing clothes matching the victim's description. The police did not detain them. The victim picked out Alejandro from his photograph in a mug book. In October 1990 Gilbert Alejandro was convicted of aggravated sexual assault by a Uvalde County jury. He was sentenced to 12 years in prison. Prosecutor's evidence at trial. The prosecution based its case on several points:  The victim identified Alejandro from a police mug shot.  The victim identified Alejandro in court (although she stated that she had a pillow over her head during the assault).  Fred Zain, the chief forensic expert for BexarCounty, Texas, testified that a DNA test of Alejandro's sample matched DNA found on the victim's clothing "and could only have originated from him [Alejandro]."  Alejandro's only alibi was from his mother, who testified that he was at home at the time of the assault.

27 Postconviction challenges Bexar County performed the forensic laboratory work in this case for the Uvalde County prosecutor's office. Bexar County discovered that the State's forensic expert in this case, Fred Zain (see also the Gerald Wayne Davis, William O'Dell Harris, and Glen Woodall cases), had falsified results and lied about his credentials when he was employed as a State police serologist in West Virginia. When Alejandro's lawyers were informed of this, they filed a writ of habeas corpus. At this time, Alejandro was released to his parents and placed on electronic monitoring. On July 26, 1994, a Uvalde County District Court heard Alejandro's petition. Present at this hearing were an original trial juror, the original jury foreman, and a Bexar County forensic DNA analyst. The two jurors testified that they based their guilty verdict solely on Zain's testimony and without his testimony the jury would have acquitted on the basis of reasonable doubt. The DNA analyst testified that results from at least one other DNA test had excluded Alejandro. He also testified that the test to which Zain testified was inconclusive and could not have been the basis of a conviction. DNA results. In July 1990 the original DNA tests done in this case—the ones Zain testified were inculpatory— were inconclusive. A Restriction Fragment Length Polymorphism (RFLP) test performed by the Bexar County crime laboratory on October 3, 1990, excluded Alejandro as the source of the semen left on the victim's nightgown. The district court also reported that an additional test was done on December 19, 1990, after the trial, and it too excluded Alejandro. According to the district court's findings of fact, Fred Zain knew of these exculpatory results and failed to report them to anyone. Conclusion. As a result of the findings of fact by the district court, the court of criminal appeals overturned Alejandro's conviction and released him to stand trial again without Zain's testimony. The district attorney, however, declined to prosecute the case. On September 21, 1994, Alejandro was released from electronic monitoring and all charges were dismissed. Alejandro served 4 years of his sentence. On June 27, 1995, he was awarded $250,000 in a civil suit against Bexar

28 12.14 STR analysis is commonly used for DNA profiling  Short tandem repeats (STRs) are genetic markers used in DNA profiling –STRs are short DNA sequences that are repeated many times in a row at the same location –The number of repeating units can differ between individuals –STR analysis compares the lengths of STR sequences at specific regions of the genome –Current standard for DNA profiling is to analyze 13 different STR sites Copyright © 2009 Pearson Education, Inc.

29 STR site 1 Crime scene DNA STR site 2 Suspect’s DNA Number of short tandem repeats match Number of short tandem repeats do not match

30 12.16 RFLPs can be used to detect differences in DNA sequences  Single nucleotide polymorphism (SNP) is a variation at one base pair within a coding or noncoding sequence  Restriction fragment length polymorphism (RFLP) is a variation in the size of DNA fragments due to a SNP that alters a restriction site –RFLP analysis involves comparison of sizes of restriction fragments by gel electrophoresis Copyright © 2009 Pearson Education, Inc.

31 Crime scene DNA Suspect’s DNA

32 Pedigree ex


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