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Chapter 19 – Molecular Genetic Analysis and Biotechnology
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Recombinant DNA technology
• One molecule composed of two distinct DNA sources
• Biotechnology– Development of commercial products; medical
applications
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Restriction endonucleases/ enzymes
• Make double-stranded cuts in DNA
• Bacterial source – guards against viral invasion– Bacterial DNA is methylated; viral unmethylated
• Name of enzymes is an abbreviation of bacterial source
• Usually recognizes 4-6 pallindromic sequences
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Digestion• Blunt ends
– Cut both strands of DNA at same location
• Sticky/cohesive ends– Produce staggered cuts; single
stranded “sticky” ends– Any DNA cut with the same
enzyme will have ends with the same sequence
• Can combine DNA from different sources and seal cuts with enzyme ligase
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Gel electrophoresis• Porous gel made of agarose or polyacrylamide
• Sample DNA mixed with loading dye that allows for visualization and increases density
• Negatively-charged DNA runs toward positive pole when electrical current passes through the gel
• Separates fragments based on size– Smaller fragments migrate the furthest – bottom of
the gel
• Ladder or marker contains fragments of known sizes to aid in determination of sample fragment size
• Expose gel to dye– Methylene bue – light box– Ethidium bromide – UV light
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Southern blotting - DNA• Restriction digestion of
genomic DNA and separated by gel electrophoresis– Large number of band sizes
produce smear on gel
• Fragments are denatured into single-strands and transferred from gel to a thin nylon or nitrocellulose membrane
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Southern blotting con’t• Membrane is exposed to
probe that has been radioactively- or fluorescently labeled– Probe has complementary
sequence to target sequence
• Unbound probe is rinsed away and bound probe is detected
• Northern blotting – RNA
• Western blotting - protein
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Cloning genes
• Produces duplicate copies of specific genes– Provides large number of copies
• Insert gene of interest into bacterial cells for rapid replication
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Cloning vector• DNA gene of interest is
inserted into a cloning vector
• Requirements:– Origin of replication– Unique restriction site
• Has only one recognition site
– Selectable marker• Antibiotic resistance
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LacZ
• Intact plasmid– Ampillicin resistance– Β-galactosidase cleaves
X-gal and bacteria is blue
• Recombinant plasmid– Ampillicin resistance– Inserted sequence
disrupts β-galactosidase gene; bacteria remains white
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Expression vectors• Used not just for
copies of gene, but to make gene product– Gene expression
• Requires sequences for transcription/ translation
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Cloning vectors • YACs
– Yeast artificial chromosomes– Yeast origin of replication, centromere, telomeres– ~600kb – 1,000kb
• BACs– Bacterial artificial chromosomes– ~100-500kb
• Shuttle vectors– Can be transferred between two different species
(bacteria and yeast)– Origin of replication and markers must be recognized by
both organisms
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Polymerase Chain Reaction (PCR)• Amplifies DNA fragments in vitro
• Taq polymerase– Isolated from hot spring bacteria Thermus
aquaticus– stable at near boiling temperatures
• Automated thermocyclers– Computer aided machine that rapidly changes
temperature
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PCR needed components• Target DNA
• Primers – 2 different (one for each strand)– Complementary to end sequences
• dNTPs
• Buffer/Mg ions
• Polymerase
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PCR steps• Denaturation
– Separates DNA into single strands
– ~90°C
• Annealing– Primers complementary pair to
DNA strands– ~55°C
• Elongation/extension– Polymerase adds new
nucleotides to primers’ 3′ end– ~72°C
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PCR con’t• Produces billions of copies of target DNA in a few hours
• Reverse transcription PCR– Makes cDNA from RNA template
• Real-time PCR– Quantifies amount after each cycle– Allows measurement of mRNA; amount of gene expression
• Limitations– Need to know DNA sequence – at least the ends– Contamination gets amplified as well– Taq polymerase has no proofreading capabilities
• Newer polymerases do
– Limited to small sizes (less than 2,000kb)
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Gel Electrophoresis Results
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Restriction Fragment Length Polymorphisms (RFLPs)
• Variation from individual to individual
• Helps with linkage studies for gene mapping
• DNA fingerprinting – Also uses microsatellites
– short tandem repeats• Size of fragment depends
on number of repeats
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DNA sequencing • Dideoxy sequencing
• Normal nucleotides dNTPs – deoxyribonucleoside triphosphate
• ddNTPs – dideoxyribonucleoside triphosphate– Missing the oxygen at the 3′
carbon– No nucleotide can be added
to strand
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DNA sequencing con’t
• 4 reaction tubes are set up – one for each base
• DNA is then denatured and run on a gel
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DNA sequencing con’t
• Sequence on gel is complementary to original strand
• Automated sequencers use ddNTPs labeled with fluorescent dye– Sample is analyzed by a
computer and sequence is graphed out
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Applications• Pharmaceuticals
– Bacterial production of human insulin, growth hormone
• Bioremediation– Bacteria genetically engineered to break down
toxic chemicals
• Agriculture– Viral/pesticide resistance; increase nutritional
value