Microbiology Biotechnology And Recombinant Dna

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Restriction enzymes

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Naturally occurring enzymes that cut DNA into fragments (palindromic-"RADAR”; “TOO HOT TO HOOT”)

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Gel electrophoresis

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a)Used to separate DNA fragments according to size b)DNA put into gel and gel electrically charged c)gel must be stained ethidium bromide to view DNA

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Vectors

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a)Small DNA circular molecule (plasmid) b)possess cut site and selectable marker (eg antibiotic gene) c)origin of replication

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DNA probes

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a)Used to locate nucleotide sequences in DNA or RNA b)Probe is single-stranded piece of DNA tagged with detectable marker (eg fluorecent light)

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Primers

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a)single-stranded DNA fragments that bind sequences of DNA b)Used in in vitro DNA synthesis

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DNA sequencing

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A process by which the sequence of nucleotides along a strand of DNA is determined

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Polymerase chain reaction (PCR)

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Creates millions of copies of given region of DNA in matter of hours (uses primers and gel electrophoresis)

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DNA Lygase

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covalently joins restriction fragments (it’s the glue)

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Recombinant Molecule

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Vector (plasmid) + Gene (dna)

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in vitro

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made to occur in a laboratory vessel or other controlled experimental environment rather than within a living organism or natural setting

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Application of Genetic Engineering

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a)protein production (insulin, hepatitis B vaccine) b)dna production (research, vaccine) c)researching gene function and regulation (research in certain bacteria and gene fusion)

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Gene Cloning

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a)coping DNA b)uses recombinant molecule (plasmid+genomic fragment)

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transgenic organism

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Plants or animals that receive engineered gene (eg pest/herbicide resistant, improved nutrient value)

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Colony blotting

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Used to detect specific DNA sequences in colonies grown in agar plates (colonies transferred to nylon membrane)

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Southern blotting

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Uses probes to detect DNA sequences in restriction fragments separated using gel electrophoresis

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Fluorescence in situ hybridization (FISH)

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Uses fluorescently labeled probe to detect certain nucleotide sequences

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DNA microarray technologies

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DNA arrays are solid supports with fixed patterns of different single-stranded DNA fragments attached

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Dideoxychain termination

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Incorporation causes DNA sequencing chain termination (fluorescent dye and gel electrophoresis used)

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Applications for DNA Sequencing

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Knowing DNA sequence of particular cell helps identify genetic alterations that can lead to sickle cell anemia or cystic fibrosis)

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PCR amplification cycle

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a)double-stranded DNA denatured by heat (95C) b)primers anneal to complementary sequence of target DNA and DNA synthesis occurs with heat- stable DNA polymerase c)duplication of target DNA

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Dideoxynucleotides

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lack 3' OH