Very Basic Biotechnology Supplemental instruction Designed by Pyeongsug Kim ©2010 Fall 2010 For Dr. Wright’s Bio 7/27 Class

Biotechnology review Define: recombinant DNA technology; genetic engineering; gene cloning How 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, © The use of microbiological and biochemical techniques to solve practical problems and produce more useful(desirable) products. Picture from Genetic 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, © From Human embryos photographed developing in vitro

Picture from 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

Recombinant DNA technology : DNA molecule created by joining DNA fragments from two different sources (two different organisms). Picture from Designed by Pyeongsug Kim, ©2010

________________ -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 Designed by Pyeongsug Kim, © Originally the bacterial enzyme that stops viral reproduction by cleaving viral DNA.

Expression vectors Picture from _______________ -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

Picture from Gene 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

Cloning 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

To express the protein, the plasmid must be placed inside a cell, either prokaryote or eukaryote. Designed by Pyeongsug Kim, ©2010

Is there my interesting DNA? ___________ -Single-stranded piece of DNA tagged with a detectable marker its complement -used to detect its complement. -Analysis of DNA within the cell (germline) DNA Probes A lineage of cells from which gametes are derived. Also known as germ track Picture from Designed by Pyeongsug Kim, ©2010

______________________ -Technique used to exponentially amplify specific regions of a DNA molecule. (PCR)Polymerase chain reaction Picture from Designed by Pyeongsug Kim, ©2010

______________________ -Can be used to detect the presence of DNA sequence. -Diagnostics, forensic analysis Polymerase chain reaction (PCR) Designed by Pyeongsug Kim, ©2010