Chapter 17 - DNA Technology Flashcards
“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.
“Transformants”
Cells that successfully obtained a vector
How can one differentiate between transformants and non-transformants?
Selective media: transformant growth is favoured over non-transformant growth (ex.
T/F: Cosmids do not exist in nature
TRUE
“Artificial chromosomes”
150-200 kb
Used for large DNA inserts
Bacterial artificial chromosomes (BACs)
Yeast artificial chromosomes (YACs)
Most stable within cell
“Genomic library”
Collection of clones that contain fragments which rep complete genome of an organism. Used when a gene of interest is on a chromosome that has not yet been sequenced.
How does one screen the genomic library?
Take genomic DNA, cleave lots of pieces and incorporate into a lot of plasmids. Hopefully, that one desired gene w/ the advantage will be ID’d.
T/F: S. cerivisiae is the most common procaryotic host of recombinant DNA.
FALSE: It’s E. coli
Describe method of “electroporation”
Cells mixed w/ recomb DNA and exposed to brief pulse of high-V electricity which renders cell membrane temporarily permeable. Then comes DNA intro into euk
“Gene gun”
Fast speed particle delivery system (metal coated w/ plasmid DNA)
“Ti plasmid”
Turmour-inducing plasmid specifically for plants
“Heterologous genes”
Cloned genes in a new host cell, may not be expressed unless they’re modified (activated)
Recomb. gene must have promoter that RNA polymerase recognizes. Introduced euk genes must be modified to have prok leader seq and all introns removed.
