Recombinant DNA recombinant DNA DNA cloning gene cloning

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1 DNA Technology

2 Recombinant DNA In recombinant DNA, DNA from two different sources, often two species, are combined into the same DNA molecule. DNA cloning permits production of multiple copies of a specific gene or other DNA segment. To work directly with specific genes or to make large amounts of gene products, scientists prepare gene-sized pieces of DNA in identical copies, a process called gene cloning.

3 Bacterium Gene inserted into plasmid Cell containing gene of interest Bacterial chromosome Plasmid Recombinant DNA (plasmid) Gene of interest Plasmid put into bacterial cell DNA of chromosome Recombinant bacterium Host cell grown in culture to form a clone of cells containing the cloned gene of interest Copies of gene Gene of interest Protein expressed by gene of interest Protein harvested Basic research on gene Basic research and various applications Basic research on protein 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

4 Restriction Enzymes Bacterial restriction enzymes cut DNA molecules at DNA sequences called restriction sites. A restriction enzyme usually makes many cuts, yielding restriction fragments. The most useful restriction enzymes cut DNA in a staggered way, producing fragments with sticky ends that can bond with other DNA fragments. DNA ligase seals the bonds between restriction fragments.

5 Restriction site DNA 5 5 Restriction enzyme cuts the sugar-phosphate backbones at each arrow. Sticky end DNA fragment from another source is added. Base pairing of sticky ends produces various combinations. Fragment from different DNA molecule cut by the same restriction enzyme Animation: Restriction Enzymes One possible combination DNA ligase seals the strands. Recombinant DNA molecule

6 Polymerase Chain Reaction (PCR) Used to produce many copies of a specific target segment of DNA. A three-step cycle heating, cooling, and replication brings about a chain reaction that produces an exponentially growing population of identical DNA molecules.

7 5 Target sequence Genomic DNA 5 Denaturation: Heat briefly to separate DNA strands 5 5 Cycle 1 yields 2 molecules Annealing: Cool to allow primers to form hydrogen bonds with ends of target sequence Primers Video: PCR Song Extension: DNA polymerase adds nucleotides to the end of each primer New nucleotides Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence

8 Gel Electrophoresis Indirect method of rapidly analyzing and comparing genomes. Uses a gel as a molecular filter to separate nucleic acids or proteins by size by exposing them to an electrical field. The phosphate groups in the sugar-phosphate backbone of DNA are negatively-charged.

9 In gel electrophoresis, the molecules move through the gel at different rates, determined largely by their size and charge. Shorter molecules move through the gel faster than longer molecules. Cathode Mixture of DNA molecules of different sizes Longer molecules Power source Gel Glass plates Shorter molecules Anode Animation: Analyzing DNA Fragments Using Gel Electrophoresis

10 In restriction fragment analysis, DNA fragments produced by restriction enzyme digestion of DNA molecules are sorted by gel electrophoresis. This is useful for comparing two different DNA molecules, such as two alleles for a gene. Normal β-globin allele 175 bp 201 bp Large fragment Ddel Ddel Ddel Ddel Sickle-cell mutant β-globin allele 376 bp Large fragment Ddel Ddel Ddel Ddel restriction sites in normal and sickle-cell alleles of β-globin gene Large fragment Normal allele Sickle-cell allele 201 bp 175 bp 376 bp Electrophoresis of restriction fragments from normal and sickle-cell alleles

11 DNA Fingerprinting An individual s unique collection of DNA restriction fragments, detected by electrophoresis and nucleic acid probes. DNA Fingerprinting