Colonies with recombinant plasmids in which lacz is

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hydrolyze X-gal. Colonies with recombinant plasmids, in which lacZ is disrupted, will be white, because they cannot hydrolyze X-gal. By screening the white colonies with a nucleic acid probe (see Figure 20.5), researchers can identify clones of bacterial cells carrying the gene of interest. Colony carrying non- recombinant plasmid with intact lacZ gene Bacterial clone Colony carrying recombinant plasmid with disrupted lacZ gene Recombinant bacteria Gene Cloning
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Uses Forensic science Agriculture Medicine vaccines, disease environment
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Test for expression of many genes at once DNA microarray assays Can reveal patterns of gene expression in different kinds of cells DNA Microarrays 1 mRNA isolated Reverse transcriptase and fluorescent DNA nucleotides 2 cDNA made from mRNA 4 Unbound cDNA rinsed away 3 cDNA applied to wells DNA microarray Each well contains DNA from a particular gene Actual size (6,400 genes) Nonfluorescent spot Fluorescent spot cDNA DNA of an expressed gene DNA of an unexpressed gene
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Electrophoresis DNA can be separated based on size and charge Eg. Phosphates are negatively charged DNA is placed in a gel and electricity is run through
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Electrophoresis Negative DNA moves toward the positive end Smaller fragments move farther and faster
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17 Electrophoresis
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Defendant’s blood (D) Blood from defendant’s clothes Victim’s blood (V) D Jeans shirt V 4 P g 8 P g Restriction Enzymes Cut human genome at distinct DNA sequences Produces a DNA fingerprint
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Copying DNA Polymerase Chain Reaction Also called PCR A method of making many copies of a piece of DNA
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Copying DNA DNA sample DNA is heated to separate two strands Cooling - DNA polymerase (Taq) is used to replicate the strands Bases / primers present Cycle can be repeated 30 -40 times
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PCR Large amounts of DNA can be made from a small starting sample
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Gene Therapy alteration of gene potential for treating disorders of a single defective gene Uses various vectors for delivery of genes Eg. retrovirus Bone marrow cell from patient Retrovirus capsid Viral RNA Cloned gene (normal allele, absent from patient’s cells) 2 Insert RNA version of normal allele into retrovirus. 1 Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. 2 Viral DNA carrying the normal allele inserts into chromosome. 3 Inject engineered cells into patient. 4
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Uses - Problems Large -scale production of human hormones Production of safer vaccines Curing of genetic -based diseases Technology not there yet
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Genetic Engineering - Agriculture Is being used to improve agricultural productivity and food quality Can insert genes for: Larger plant size Better flavour of plant Better coats Leaner meat Faster maturation
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APPLICATION Genes conferring useful traits, such as pest resistance, herbicide resistance, delayed ripening, and increased nutritional value, can be transferred from one plant variety or species to another using the Ti plasmid as a vector.
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