Ch 12

Lac Operon 0Kh4&feature=relatedhttp:// 0Kh4&feature=related AUJd0&feature=relatedhttp:// AUJd0&feature=related RNA Splicing 5R8yY&feature=relatedhttp:// 5R8yY&feature=related BGw_pQ&feature=related

Recombo DNA cD2g&feature=relatedhttp:// cD2g&feature=related PCR woZKU&feature=related

Researchers can insert desired genes into plasmids, creating recombinant DNA and insert those plasmids into bacteria Bacterium Bacterial chromosome Plasmid 1 isolated 3 Gene inserted into plasmid 2 DNA isolated Cell containing gene of interest DNA Gene of interest Recombinant DNA (plasmid) 4 Plasmid put into bacterial cell Recombinant bacterium 5 Cell multiplies with gene of interest Copies of protein Copies of gene Clone of cells Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein used to dissolve blood clots in heart attack therapy Protein used to make snow form at higher temperature Figure 12.1

Creating recombinant DNA using restriction enzymes and DNA ligase Restriction enzyme recognition sequence G A A T T C C T T A A G DNA C T T A A A AT TC G C T T A A Addition of a DNA fragment from another source Two (or more) fragments stick together by base-pairing G A AT T C C T TA A G G A AT T C C T TA A G 5 DNA ligase pastes the strand Restriction enzyme cuts the DNA into fragments Recombinant DNA molecule G G Sticky end G Figure 12.2

Cloning a gene in a bacterial plasmid 1 Isolate DNA from two sources 2 Cut both DNAs with the same restriction enzyme E.coli Plasmid DNA Gene V Sticky ends 3Mix the DNAs; they join by base-pairing 4 Add DNA ligase to bond the DNA covalently 5 Put plasmid into bacterium by transformation Gene V Recombinant DNA plasmid Recombinant bacterium 6 Clone the bacterium Bacterial clone carrying many copies of the human gene Human cell Figure 12.3

Cell nucleus Isolation of mRNA and addition of reverse transcriptase; synthesis of DNA strand RNA splicing 2 Transcription 1 3 Breakdown of RNA 4 Synthesis of second DNA strand 5 mRNA DNA of eukaryotic gene IntronExon RNA transcript ExonIntronExon Reverse transcriptase Test tube cDNA strand being synthesized cDNA of gene (no introns) Reverse transcriptase makes single stranded DNA from mRNA cDNA

Recombinant cells and organisms can mass-produce gene products Table 12.6

Therapeutic hormones & vaccines –In 1982, humulin, human insulin produced by bacteria Became the first recombinant drug approved by the Food and Drug Administration –Vaccines are harmless mutants or derivative of a pathogen that stimulate the immune system Figure 12.7A

GM organisms –Acquired genes by artificial means –Transgenic organisms Agrobacterium tumefaciens DNA containing gene for desired trait Ti plasmid Insertion of gene into plasmid Recombinant Ti plasmid 1 Restriction site Plant cell Introduction into plant cells 2 DNA carrying new gene Regeneration of plant 3 Plant with new trait

Gene therapy (or the alteration of an afflicted individual’s genes) may someday help treat a variety of diseases Cloned gene (normal allele) 1 Insert normal gene into virus 2 Infect bone marrow cell with virus 3 Viral DNA inserts into chromosome 4 Inject cells into patient Bone marrow Bone marrow cell from patient Viral nucleic acid Retrovirus Figure 12.13

Cycle 1 yields 2 molecules Genomic DNA Cycle 3 yields 8 molecules Cycle 2 yields 4 molecules Target sequence Heat to separate DNA strands Cool to allow primers to form hydrogen bonds with ends of target sequences PrimerNew DNA 5 DNA polymerase adds nucleotides to the 3 end of each primer 5 PCR used to amplify target section of DNA

Gel electrophoresis sorts DNA molecules by size ++ – – Power source Gel Mixture of DNA molecules of different sizes Longer molecules Shorter molecules Completed gel Figure 12.10

STR –Short sequences of DNA repeated many times in a row –STR analysis compared lengths of STR sequences at specific sites on the genome

How Restriction Fragments Reflect DNA Sequence –Restriction fragment length polymorphisms (RFLPs) reflect differences in the sequences of DNA samples Crime sceneSuspect w x yy z Cut DNA from chromosomes C C G G G G C C A C G G T G C C C C G G G G C C C C G G G G C C Figure 12.11A

–After digestion by restriction enzymes the fragments are run through a gel – + Longer fragments Shorter fragments x w y z y 12 Figure 12.11B

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

Used in forensic investigations 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

DNA fingerprinting can help solve crimes Defendant’s blood Blood from defendant’s clothes Victim’s blood Figure 12.12A Figure 12.12B