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22 Cards in this Set
- Front
- Back
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recombinant DNA technology |
-goal is to combine DNA from 2 sources -previously examined phenotypic difference but now we have ability to analyze nucleotide sequences -used to create hundreds of commercial products, drugs, hormones, enzymes, crops -biotechnological industry |
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problems working at a molecular level |
-extremely small -no physical mark for the beginning or end of gene -gene of interest is incorporated into entire genome (need to isolate gene) -linear DNA fragments degrade quickly (have to be inserted into a stable form) -techniques are often inefficient (need to produce millions of copies) |
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restriction enzymes |
-restriction enzymes: endonucleases that are used to cut DNA strands at specific sites -specific sites: usually palindromic sequences 4-6 bp long -cuts can be staggered or blunt |
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staggered vs blunt ends |
-HindIII vs PvuII -DNA molecules exposed to same restriction enzyme have complimentary sticky ends that pair if fragments are mixed |
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gel electrophoresis |
-after splicing the DNA fragments, used to separate fragments and estimate sizes -made of agarose gel that forms after boiling (like Jell-O) -gel is submersed in a low-salt buffer to allow electricity to go through the gel and maintain the pH -DNA is negative so it migrates from anode --> cathode -small fragments more quicker than larger -visualize by staining the gel with specific dyes which become fluorescent under UV light, or by radioactively labelling and detecting using autoradiography X-rays |
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gene cloning |
-placing a fragment of DNA in a bacterial cell and allowing the cell to replicate -produces identical copies (clones) of the DNA -use a vector |
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vectors |
-origin of replication: ensures replication inside the host cell -selectable markers: enable cells containing the vector to be identified -unique restriction sites: recognized by restriction enzymes, allow it to be inserted into host cell -can be: plasmids, cosmids, or artifical chromosomes |
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plasmids |
-3-5kb circular DNA molecules -optimized for lab experiments -once a gene is placed inside a plasmid, it must be introduced into host cell -usually done via transformation -host cell divides and copies of recombinant plasmids can be detected by selectable markers: usually antibiotic resistance (e.g. lacZ gene) |
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lacZ gene |
0contains many restriction sites which allow easy insertion -in absence of foreign fragment, lacZ gene is active and produces B-galactosidase (lacZ+) -if foreign DNA is inserted, B-galactosidase is not produced (lacZ-) -can plate bacteria on a medium containing ampicillin to determine if bacteria contains a plasmid or not |
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PCR |
-polymerase chain reaction -revolutionized molecular genetics -most widely used tool in molecular biology -uses heat-stable DNA polymerases (Taq polymerase), primers, dNTPs, Mg++, buffer, and salt -applications: DNA fingerprinting, introduce new traits, identify genetic variation within a population |
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PCR steps |
1. denaturation: melting the two DNA strands by heating to 94 degrees (dsDNA --> ssDNA) 2. primer annealing: cool the reaction to between 50-70 degrees, marked the region that is going to be amplified, designed by the researcher 3. primer extension: at 72 degrees using heat-stable polymerase (Taq), new nucleotides are brought in -exponential amplification |
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gene libraries |
-collection of cloned sequences
-because cloned DNA segments are small, may represent only a single gene or portion of a gene -need many clones to cover an organism's genome -two types: 1. genomic: contains ALL the DNA sequences found in the genome 2. cDNA (complementary DNA): consists only of DNA sequences that are transcribed into mRNA -advantage: bacteria don't have ability to remove introns, so when trying to produce eukaryotic protein in bacteria cDNA eliminate introns so it doesn't interrupt cloned sequences |
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sequencing DNA |
-molecular tool used to determine the sequence of bases -provides information about gene structure/function -used to compare individuals/species -1970s: Sanger method relied on DNA replication but with incorporation of modified nucleotides that prevent further synthesis; used polyacrylamide gel so difference in length could be seen |
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original Sanger sequencing |
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current Sanger sequencing |
-still incorporates modified nucleotides stopping replication |
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products of biotechnology |
-Pharmaceutical Products: insulin for diabetic, human growth hormone for children with growth deficiencies, clotting factors for hemophiliacs -Specialized bacteria: breakdown toxic pollutants, enhance oil recovery, increase nitrogen uptake by plants -Agricultural products: GMO’s, pesticides -Gene therapy agents: treating human diseases such as cancer, heart disease, AIDS; currently only using somatic cells; gametic cells, ethical to alter generations to come? |
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concerns about recombinant DNA |
-unnatural process (ethical concerns) -messing with genomes and don’t know long-term consequences (effect on individual organisms) -escape of transgenes into ‘wild’ species may create invasive microbes/plants/animals (risk to ecosystem) |
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genomics |
-attempts to understand the content, organization, function, and evolution of genetic information -three types: 1. structural: determines organization of sequences within genome 2. functional: determines function of sequences found by structural genomics 3. comparative: compares gene content/function/organization between different organisms |
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genetic apping |
-provide rough estimates of locations of genes relative to other genes -based on recombination -ultimate goal = determine order of nucleotides in entire genomes -two types: 1. map-based sequencing 2. whole genome shotgun sequencing |
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map-based sequencing************* |
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whole genome shotgun************* |
-smaller fragments than map-based -more DNA than map-based -put it all together to sequence organism's entire DNA |
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human genome project |
-1990, 15 years and $3 billion estimate -used map-based sequencing -1998, Craig Venter founded Celera to privately sequence using shotgun -both projects released in 2000s -provides tools for detecting and mapping genetic variants --> used to study diseases/tmts |
