Chapter 8 Recombinant DNA Technology Flashcards
Biotechnology
the use of microorganisms to make practical products
Recombinant DNA Technology
intentionally modifying genomes of organisms for practical purposes
3 goals of Recombinant DNA Technology
1 eliminate undesirable phenotypic traits
2 combine beneficial traits of 2 or more organisms
3 create organisms that synthesize products humans needs
5 tools of Recombinant DNA Tech
1 mutagens 2 reverse transcriptase 3 restriction enzymes 4 vectors 5 gene libraries
Mutagens
-physical + chemical agents that produce mutations in DNA
Mutagens for Recombinant DNA
- used to create changes in microbe’s genomes to change phenotype
- selects for + cultures cells w beneficial characteristics
- mutated genes alone can be isolated
Reverse Transcriptase
an enzyme that uses RNA template to transcribe a molecule of cDNA
(turns RNA to DNA)
Reverse Transcriptase for Recombinant DNA
- isolated enzymes fr retrovirus
- easier to isolate mRNA for desired protein
- cDNA generated from mRNA of eukaryotes has intron removed to allow prokaryotic cells to produce eukaryotic proteins
removal of introns from cDNA from eukaryotic mRNA. This allows for…..
prokaryotic cells to produce eukaryotic proteins
cDNA
complimentary DNA
Restriction Enzymes/Endonucleases
- bacterial enzymes that cut DNA molecs only at restriction sites
- creates recombinant DNA by joining fragments
2 types of Restriction Enzymes/Endonucleases
1 cuts w sticky ends
2 cuts w blunt ends
-categories are based on type of cut
Vectors
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nucleic acid molecules that deliver a gene into a cell
-includes viral genomes, transposons, and plasmids
Gene Libraries
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- collection of bacterial or phage clones
- each clone in library often contains one gene of an organism’s genome
- provides a ready source of genetic material
Polymerase Chain Reaction [PCR]
- multiplying DNA in vitro (like a xerox machine)
- large number of molecs of DNA are produced
- can be automated using a thermocycler
3 repetitive steps of PCR
1 denaturation
2 priming
3 extension
why is it important to be able to amplify DNA?
- epidemiologists use to amplify genome of unknown pathogen
- amplified DNA from Bacillus anthraces spores in 2001 to identify source of spores
2 methods of inserting DNA into cells
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1 Natural Method
2 Artificial Method
Gene Gun + Microinjection
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-artificial method of injections
Gene Mapping
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- locating genes on a nucleic acid molecule
- provides useful facts concerning metabolism, growth characteristics, and relatedness to others
Nucleotide Sequencing
- used to relate DNA sequence to protein functions
- Genomics
- elucidation of the genomes of pathogens is a priority
Genomics
sequencing and analysis of the nucleotide bases of genomes
Protein Synthesis
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creation of synthetic proteins by bacteria and yeast cells
Vaccines
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- production of safe vaccines
- subunit vaccines
new approaches to stimulate immunological memory
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- intro of genes of pathogens into fruits + veggies
- injecting humans w plasmid carrying gene fr pathogen
- –human synth pathogen’s proteins
Gene Screening
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- DNA microarrays are used to screen individuals for inherited disease caused by mutations
- can also ID pathogen’s DNA in blood or tissues
Gene Therapy
- missing or defective genes are replaced w normal copies
- some patient’s immune system react negatively
- treatment of cancer by directing toxin-loaded virus to the cancer cells
DNA fingerprinting
ID individuals or organisms by their unique DNA sequence
Gene Therapy for killing cancer
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TERT gene product> Telomerase
-hTERT promoter is active in all types of tumors and immortal cells but inactive in somatic tissues
Telomerase
- highly active enzymes that divide rapidly in cells
- abnormally active in most cancer cells which grow + divide w/o control or order
Sticky ends
able to pair w other complimentary ends
Blunt ends
ligase brings blunt ends together
E.Coli DNA polymerase vs. Thermus aquaticus (TAQ) DNA polymerase
- TAQ DNA polymerase is able to withstand protein-denaturing conditions (high heat), whereas E.Coli is not
- E.Coli was originally used for PCR but has been replaced w TAQ
- used for DNA aplification
Gel Electrophoresis
uses electrical charge to separate molecules according to their size
-separates DNA fragments by size
reverse transcriptase is found in which organism ?
certain RETROVIRUSES like HIV virus
