Biotechnology Spring 2013 - Althoff Reference: Mader & Windelspecht Ch. 14) Lec 21 DNA Cloning Recombinant DNA PCR Analysis Biotechnology.

BiotechnologySpring 2013 - Althoff Reference: Mader & Windelspecht Ch. 14)

Lec21

• DNA Cloning• Recombinant DNA• PCR • Analysis• Biotechnology

• By definition, __________ is the production of genetically __________ copies of DNA, cells, or organisms through some __________ means.

• Ex.: when a plant sends up an underground stem or a root sends up a new shoot, the resulting plant is a clone of the original plant.

• Ex.: members of a bacteria colony on petri dish are clones because the all came from the division of a single original cell

DNA Cloning

• Reasons to clone:a) determine the difference in base _________ between a normal gene and a mutated gene b) use the genes to genetically _______ organisms in a beneficial way

• When cloned genes are used to modify a human, the process is called ______________

• Otherwise, organisms are called ___________ organisms (trans = across, genic = producing).

• Value of transgenic organisms – produce a product desired by humans

DNA Cloning

• Recombinant DNA = ________

• Contains DNA from ___________ different sources, such as a human cell and a bacterial cell.

Recombinant DNA Technology

• To make rDNA, one needs a _________ (i.e., a carrier) by which the rDNA will be introduced into a host cell

• Example of a vector: ________ = small accessory rings of DNA found in ________ that were first discovered in E. coli. The ring in not part of the main bacterial chromosome…and replicates on its own.

• Need 2 enzymes to introduce foreign DNA into vector DNA:1) a restriction enzyme which cleaves DNA 2) DNA ligase (ligo = bind)…a ‘sealer’

Recombinant DNA Technology

1

2

• ________

• Developed by Kary Mullis in 1985.

• Mullis earned Nobel Prize for Chemistry in 1993

• Widely used in research labs to ____________ of a segment of DNA quickly in a test tube

• Process mimics DNA replication in the cell with the exception that the PCR is very specific—it amplifies (aka makes copies of) only a targeted DNA sequence

Polymearse Chain Reaction

• Requires the use of a) DNA polymerase b) supply of nucleotides for the new

strands of DNA

• The DNA polymerase used is from the bacterium Thermus aquaticus—which lives in hot springs. It can withstand high temperatures used to separate the double-stranded DNA

• Referred to as a chain reaction because the targed DNA is repeatedly replicated as long as the process continues.

PCR

Polymearse Chain Reaction

• DNA amplified (i.e., copies made) enables one to decipher evolutionary history of organisms by looking at mitochrondrial DNA. It can also be used to ID a corpse to match to DNA found on the bristles of a toothbrush.

• Early analysis done using a process called ____ ________________ a) a process that separates DNA fragmentsaccording to their size b) separation process leaves a distinctive band that identifies a person, for example

Analyzing DNA

• Gel electrophoresis still used widely today…including in your Genetics course (if you continue on in Biology)

• More advanced (and requiring much more expensive equipment) is the STR Profiling method = short tandem repeat profiling.

• STR does __ require use of restriction enzymes

• Newest method to produce DNA profiles or “fingerprints” accomplished by fluorescently labeling DNA fragments. Involves a laser exciting the fluorescent DNA and a detector recording the amount of emissions for each DNA fragment

Analyzing DNA…con’t

DNA “Fingerprint”

• Transgenic bacteria, plants, and animals often now called GMOs = genetically modified organisms.

• GMO – bacteria: promote health of plants including one that helps plant roots be protected from some insect species. Some used to decompose oil !!!!

Biotechnology Products

• Gene Therapy

• Current use and much future potential for application to _____ many conditions harmful to humans