Chapter 17 - DNA Technology
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“Recombinant DNA”
• Use enzymes + lab techniques to isolate segments of interest • Combine/splice DNA from diff species • Create genes w/ new functions • Typ. involves propogation of recombinant DNA in bact/yeast cell
“Genetic engineering”
• Use lab-based tech to alter DNA • Changing single base pair • Deleting regions DNA • Adding new segment • May involve production of desired trait • Cancer therapies, brewing yeasts, gen-mod plants/livestock
T/F: Plasmids are extrachromosomal DNA in bact, archaea, and euk
FALSE: Bact, arch and some fungi
T/F: Genes on plasmids NOT essential to host, but may confer selective advantage
TRUE
Desc method of “Restriction enzymes”
• Bind to spec sequences (recognition sites) • Cleave doub-tranded DNA at this site or defined distance from it
What is the difference between Type I/III and Type II restrict. enzy?
Type I/III: ID rec site THEN cleave DNA a dist from it Type II (most pop): cut DNA directly @ rec site
List two restriction enzymes, their microbial source, their recognition sequence and the ends that are produced.
• BamHI Source: Bacillus amyloliquefaciens H Rec seq: 5’ GGATCC ‘3 + reverse seq Ends produced: 5’ G GATCC 3’ / 3’ CCTAG G 5’ • EcoRI Source: Escherichia coli Rec seq: 5’ GAATC 3’ + reverse seq Ends produced: 5’ G AATTC 3’ / 3’ CTTAA G 5’
What is challenge w/ cloning euk DNA into bacteria?
Pre-mRNA (hnRNA) requires removal of introns, add. of poly A tail, 5’ cap (need to use version that can be made in bact)
What is solution to cloning euk DNA into bact?
Construction of complementary DNA (cDNA) involving role of key enzyme: reverse-transcriptase (uses euk mRNA as a template for cDNA, w/ primer TTTTTT…)
T/F: Positively-charged DNA moves towards the negative pole of the gel during gel electrophoresis.
FALSE: NEGATIVELY-charged DNA moves towards the POSITIVE pole.
Describe the mechanism by which gel electrophoresis is used to separate DNA.
• Restriction endonucleases selectively cleave DNA segments • Restriction fragments are produced • Processed DNA samples are loaded with dye and placed in the wells of the gel • An electric current is induced in the gel • As the current flows towards the positive pole, the negative DNA fragments travel through the gel. Smaller fragments travel farther than bigger ones. • Fragments are separated based on size and compared to a ladder
Describe the southern blotting technique to ID strands of DNA.
• Separate DNA molecules • Transfer-separated DNA molecules (buffer passes upwards through layers to transfer fragments on to filter) • Filter placed on X-ray film to produce image of DNA bands (hybridize to radioactive spec DNA probe)
T/F: The radioactive probe present in the Western Blotting Technique is important for for IDing DNA sequences of interest and trying to prove with certainty that a band found on the cell is a spec gene seq.
FALSE: This is important for Southern blotting. Western Blotting concerns proteins.
What does the rxn mixture for PCR contain?
• Primers • Target DNA (including gene of interest) • Thermostable DNA polymerase (Taq) • Each of 4 deoxyribonucleotide triphosphates (dNTPs)
“Primers” for PCR?
Synthesized DNA oligonucleotides 15-30 nucleotides long which provide the 3’ OH needed for DNA synthesis during PCR
What is the instrument used for PCR?
Thermocycler
Describe the process of PCR.
• DNA is denatured • Primers anneal to target DNA • Copies of target DNA are synthesized
T/F: The PCR reaction sequence demands the annealing process be completed at the lowest temperature between “denaturing” and “synthesis”
TRUE Denaturing: 94-98ºC Annealing: 50-65ºC Synthesis: 72ºC
What are the design parameters for PCR?
• Primer MUST be unique to sequence • < 100bp in length
Compare End-point and Real Time reverse transcriptase
End-point RT-PCR: for obtaining large quant of spec DNA, products collected + purified Real-time RT-PCR: Quant # of copies fluorescent probe added to rxn mix to measure @ end of each cycle
Besides PCR, how else can DNA be synthesized?
Oligonucleotides (step-wise, single nucleo added @ end of growing chain)
What are the 4 types of cloning vectors?
• Plasmids • Phages + viruses • Cosmids • Artificial chromosomes
Each type of cloning vector generally has:
• An origin of replication • Selectable marker (eg. AmpR gene for Ampicillin resistance) • Multicloning site/polylinker (region w/ unique restrict. sites)
T/F: If a new gene is inserted into a specified DNA sequence, then the sequence will still be able to encode - the inserted gene is dormant.
FALSE: Gene will no longer be able to encode.