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Recombinant DNA (rDNA)
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-DNA containing genes from another source
-new genes can give cell new capabillities
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Biotechnology
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-uses recombinant DNA to "program" microbes
-uses: -produce products (enzymes, vaccines, insulin etc..)
- give cells special properties (frost resistance etc)
-treat diseases
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Tools of Biochnology
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-restriction enzymes
-PCR (polymerase chain reaction)
-gel electrophoresis
-DNA sequencing
-vectors
-DNA probes
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Restriction enzymes
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-bacterial enzymes that cut DNA
-cuts @ specific base sequence
-leaves "sticky ends", single stranded ends
-allows genes to be inserted into plasmids
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PCR (polymerase chain reaction)
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-makes billions of identical copies of DNA strand
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To prepare PCR
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-prepare solution:
-primers
-free nucleotides
-high-temp. DNA polymerase
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After preparing PCR soulution
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-place solution into thermalcycler
-Heat to seperate DNA into single strands
-Cool to allow new nucleotides to attach
(# of strands doubles)
- Repeat cycle 20-30 times
(billions of copies in a few hours)
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Gel Electrophoresis
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-Place DNA into wells of agar gel
-Turn on current
-DNA has a (-) charge and migrates from (-) to (+) end, smallest pieces move fastest (travel farthest)
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DNA sequencing determines
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-determines sequence of bases in the DNA
-reveals location of important genes on chromosome
-Dye terminator sequencing commonly used
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Dye terminator sequencing
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-sequence sense strand only, add chain-terminating nucleotides to PCR solution (A,T, G,C)
-Ea. terminator labeled, ea. is a diff. fluorescent color (wavelength)
-Run PCR, ea strand grows until chain terminator attaches
-separate strands by length (polyacrylamide gel)
-optical scanner reads sequence
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Vectors (carrier)
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-carries desired genes (DNA segments) into cells
-once inside cell they are self-replicating (ea. new cell gets copy of genes ea. time cell divides)
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2 types of vectors
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-viruses
-plasmids
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Viruses
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-carry large DNA segments into cells
-phages carry large pieces of DNA into
bacteria
- animal cells
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Plasmids
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-carry small segments of DNA
- shuttle vectors: plasmids that can exist in several diff. species
-can carry DNA into bacteria, plant,
mammals etc.
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DNA probes
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-pieces of single stranded DNA used to find specific genes
-Base sequence is complementary to gene of interest (binds to it w / H-bonds (hybridizes)
-Has fluorescent or radioactive "tag" so it can be found
-DNA syntheses machines can produce short DNA pieces
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