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VeryVery Basic Biotechnology Basic BiotechnologySupplemental instruction
Designed by Pyeongsug Kim ©2010 [email protected]
Fall 2010For Dr. Wright’s Bio 7/27 Class
Biotechnology review Define: recombinant DNA technology; genetic engineering; gene cloningHow do restriction enzymes work? How are they used for cloning? What is an expression vector?How does the polymerase chain reaction (PCR) work?What is a probe? What are some possible uses for probes?
Designed by Pyeongsug Kim, ©2010 www.science-i.com
BiotechnologyBiotechnology-The use of microbiological and biochemical techniques to solve practical problems and produce more useful(desirable) products.
Picture from http://www.plantsci.cam.ac.uk/Haseloff/teaching/PlantBiotech/index.html
Genetic engineeringGenetic engineering
-Alternating an organism’s genetic engineering using in vitro techniques.
A procedure performed in vitro (Latin: within the glass) is performed not in a living organism but in a controlled environment, such as in a test tube or Petri dish.
Designed by Pyeongsug Kim, ©2010 www.science-i.com
From http://en.wikipedia.org/wiki/In_vitro
Human embryos photographed developing in vitro
Picture from http://employees.csbsju.edu/hjakubowski/classes/ch331/dna/plasmid.gif
Using microbes(usually bacteria)Using microbes(usually bacteria)-Protein production-DNA production-Gene regulation research-”DNA library”-Why microbes? Grow fast (even single-celled eukaryotes like yeast) Don’t take up much space Economical source of genetically-engineered material Have the same DNA as all living organisms.(DNA or RNA)
Designed by Pyeongsug Kim, ©2010 www.science-i.com
Recombinant DNA technologyRecombinant DNA technology: DNA molecule created by joining DNA fragments from two different sources (two different organisms).
Picture from http://citnews.unl.edu/hscroptechnology/html/printLesson.shtml?lessonID=990631835 Designed by Pyeongsug Kim, ©2010 www.science-i.com
________________-naturally occurring bacterial enzyme -recognizes and cut a specific nucleotide sequence of DNA.-used to cut DNA at specific points during production of recombinant DNA.-ECoRI ~ restriction enzyme from E.Coli(RY13)
-can be joined to fragments to other source.
For recombinant DNA, first, the DNA needs to be cut!And glue it!
Restriction enzymes
Picture from http://employees.csbsju.edu/hjakubowski/classes/ch331/dna/plasmid.gif Designed by Pyeongsug Kim, ©2010 www.science-i.com
Originally the bacterial enzyme that stops viral reproduction by cleaving viral DNA.
Expression vectors
Picture from http://www.artes-biotechnology.com/service/hansenula.jsp
_______________-Generally a plasmid-used to introduce a specific gene into a target cell.-facilitate transcription and translation of cloned DNA.
Designed by Pyeongsug Kim, ©2010 www.science-i.com
Picture from http://www.artes-biotechnology.com/service/hansenula.jsp
Gene cloningGene cloning -Fragments can be “cloned” into “expression vectors” to be inserted into microbes -the cloned cDNA is incorporated into an expression vector or plasmid and transferred into bacterial or yeast cells.The cells multiply (cloning)
The interesting DNA needs to be cloned.
Designed by Pyeongsug Kim, ©2010 www.science-i.com
Cloning stepCloning step -Isolate DNA. -Use a restriction enzyme to generate fragment. -Generate a recombinant molecule. -Introduce recombinant molecule. -Introduce recombinant into new host.
Designed by Pyeongsug Kim, ©2010 www.science-i.com
To express the protein, the plasmid must be placed inside a cell, either prokaryote or eukaryote.
Designed by Pyeongsug Kim, ©2010 www.science-i.com
Is there my interesting DNA?___________-Single-stranded piece of DNA tagged with a detectable marker -used to detect its its complementcomplement. -Analysis of DNA within the cell (germline)
DNA Probes
<Germline> A lineage of cells from which gametes are derived. Also known as germ track
Picture from http://citnews.unl.edu/hscroptechnology/html/printLesson.shtml?lessonID=990631835 Designed by Pyeongsug Kim, ©2010 www.science-i.com
______________________-Technique used to exponentially amplify specific regions of a DNA molecule.
(PCR)Polymerase chain reaction
Picture from http://blogs.newamericamedia.org/photo-of-the-day/103/pcr http://nobelprize.org/nobel_prizes/chemistry/laureates/1993/illpres/pcr.htmlDesigned by Pyeongsug Kim, ©2010 www.science-i.com