Recombinant DNA and Genetic Engineering Flashcards
Genetic engineering-
modifying the DNA of an organism to new genes w new traits
Biotechnology-
use of organisms to benefit humanity
Recombinant DNA technology-
researchers splice together DNA
from different organisms
steps and info recombinant DNA tech
Scientists introduce foreign DNA into the cells of microorganisms; When cell divides, the foreign DNA is replicated and transmitted to daughter cells; DNA sequence is therefore cloned to provide millions of identical copies
geneticists cut both foreign DNA and plasmid DNA with the same restriction enzyme. The two types of
DNA are mixed together and combined
Restriction enzymes
are used to cut DNA molecules only in specific places; Each enzyme recognizes and cuts the DNA at different site; Many cut at palindromic sequences; Cuts both strands, but in a staggered fashion- produces
sticky ends; If sticky ends of two molecules are put together, they are sealed by DNA ligase
origin of restriction enzymes
Derived from bacteria cells, which use these enzymes as defense against viral DNA
palindromic sequences-
base sequence of one strand reads the same as its complements when both are read in the 5’ to 3’ direction
sticky ends-
tails which can base pair with a complementary
tail of any other DNA fragment or molecule cut by the same restriction enzyme
Plasmid-
very small circle of extra DNA found in bacterial cells that has a few genes and is replicated along w the other bacterial DNA.
Transformation
DNA (plasmids) from one cell can be taken up by other bacterial cells; Useful to bacteria because it can offer resistance to antibiotics
vector
carrier capable of transporting a DNA fragment into a cell. plasmids, viruses, etc.
vector capabilities
Vector can only carry a certain size of DNA fragment
smaller than 10 kb E. coli, up to 23 kb bacteriophage
recombinant tech diagram
vector page
Genomic library-
collection of thousands of DNA fragments that
represent all the DNA in a genome
Genomic library- process
Each fragment from genomic library is inserted into a plasmid, which also
contains the gene for
antibiotic resistance
• Plasmid is then inserted into
bacterial cell
• Cells are incubated on a medium containing antibiotics, so those bacteria that do not contain the plasmid will die, while those that incorporated the plasmid will grow
• Entire genome is therefore stored in collection of
recombinant bacterial cells
Genomic library- diagram
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Chromosome library-
containing all DNA fragments of a specific chromosome
cDNA library-
contains DNA derived
from mRNA after processing- does not contain introns
cDNA
complementary DNA
cDNA diagram
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genetic probe
To find a gene of interest in a library, a genetic probe is used- radioactively labeled segment of single-stranded DNA that can hybridize, or base-pair, to complementary sequences in the target gene
genetic probe diagram
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Polymerase Chain Reaction (PCR) purpose
Can amplify a tiny sample of DNA millions of times in a few
hours (discovered in 1985)
Polymerase Chain Reaction (PCR) process
done in a thermocycler
Mix together sequence of DNA, primers, nucleotide bases, and a special polymerase called Taq polymerase which does not denature in heat
• Heat and cool mixture over and over, allowing the DNA strands to separate and copy
- DNA doubles with every cycle, so after only 20 cycles, over 1m copies are made
PCR diagram
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gel electrophoresis
method that separates different molecules using
a charged field
gel electrophoresis diagram
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gel electrophoresis process
DNA or RNA is loaded into a gel
o An electric current is applied, making one end
positive and one end negative (- by the wells)
o DNA and RNA have a slight negative charged due to
negatively charged phosphate groups- therefore they
migrate toward the positive pull
o Different fragments travel at different speeds:
Shorter fragments travel faster thru the gel
Larger fragments travel slower thru the gel
o If DNA of a known size is added, scientists can compare these to the fragments to figure out the molecular weight
DNA fingerprint-
a unique combination of DNA sequences that are inherited from parents.
tandem repeats-
some parts of DNA fingerprints
many copies of the same short base sequences, one after another ex–four repeats of TTTC in one person as compared to 15 repeats in another person.
Restriction Fragment Length
Polymorphisms (RFLPs)
tandem repeats–Restriction enzymes that cut the DNA will produce different size fragments based on the number of repeats; these different size fragments will be visible in a gel electrophoresis. The banding pattern of DNA fragments (5 – 10 RFLP regions are
used) is the DNA fingerprint. Can determine
paternity; Suspects from crime scene can be proven innocent.
Designer Plants:
application of DNA tech. Can hybridize plants with other varieties of plants in order to transfer desired genes.Use a plasmid to infect plants from the bacteria called
Agrobacterium tumefaciens- causes tumor (mass of cells) of recombinant DNA. New plants can then be regenerated from individual cells. Plasmid is therefore called Ti plasmid
ex–insert a built-in insecticide gene into cotton plants so farmers don’t have to use so many insecticides,
designer plants diagram
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DNA fingerprint diagram
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Gene therapy-
In individuals with single gene disorder, a new functional gene can be inserted into the cells Must be cells that actively divide in the body so new
gene will be replicated. Must be disease that can be cured by production of the normal protein. New gene can be inserted into cells using a harmless virus as a vector
Transgenic animals-
animals in which foreign genes have been introduced. Transgenic animals can often be engineered to produce foreign proteins of therapeutic or commercial importance. Often done in mice to study certain genes- ex- injected mouse embryo cells with gene for rat growth hormone.
Transgenic animals- creation
Produced by injecting DNA of a particular gene into the nucleus of a fertilized egg cell or nucleus of embryonic stem cells, Then the egg (or stem cells) is placed into the uterus of a female
and allowed to develop.
Transgenic animals can often be engineered to produce foreign proteins of therapeutic or commercial importance–give example
Ex- foreign gene can be inserted into gene that codes for milk proteins. So foreign gene is activated only in
mammary tissues involved in milk production- whenever
the animal (cow, goat, sheep) is milked, that protein is
produced
Human Genome Project
Using all these techniques, scientists have been able to sequence all 3
billion bases of the human genome
- Completed in 2003
- Estimated that there are about 20,000 genes, yet there are almost as many pseudogenes- inactivated, nonfunctional copies of genes- as there are genes
- Discovered that protein-coding genes make up less than 2 percent of
our genome. Millions of transposable elements repeated over and
over make up more than half of it.
Production of pharmaceutical products:
Insulin used to be derived from animals, but many diabetics were allergic to insulin from animal source. Using recombinant DNA, the gene for insulin can be spliced into E. coli–> e coli produces human insulin. Similarly, human growth hormone (only used to be available from cadavers) can also be manufactured in bacteria.
