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Chapter 20: DNA Technology and Genomics. Important Point:. If you are having trouble understanding lecture material: Try reading your text before attending lectures. And take the time to read it well!.
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Important Point: If you are having trouble understanding lecture material: Try reading your text before attending lectures. And take the time to read it well!
DNA technology is the chemical manipulation of the genotypes and resulting phenotypes of organisms such that living organisms are modified • Alternatively, no-longer-living organisms or their no-longer-living parts may be analyzed chemically at the level of genotype • DNA technology has revolutionized how scientists study the genetics, biochemistry, even the ecology and evolutionary biology of organisms • Genetic engineering is the artificial manipulation of the genetic material of organisms, including the creation of novel genetic material (i.e., novel nucleotide sequences) • Biotechnology is the development of novel biological products, indeed whole industries are now devoted to the production and analysis of biological materials DNA Technology
Clone Identification • Probing for correct DNA sequence • Probing for correct protein product • Antibiotic resistance • β-galactocidase expression • Etc. • Once you have the correct clone, then what? • Subcloning • Expression vector • Protein characterization • Protein purification • Etc. Have Transformant, then What? “The final and most difficult part of cloning a particular gene is identifying a colony containing that gene among the many thousands of colonies carrying other pieces of… DNA.” p. 388, Campbell & Reece (2005)
5’…GAATTC…3’ 3’…CTTAAG…5’ 5’…G -OHP-AATTC…3’ 3’…CTTAA -PHO-G…5’ Restriction Endonucleases • A Restriction Endonucleases will cut both strands of a DNA duplex at a specific place • These “places” need not be directly opposite: 5’…GAATTC…3’ 3’…CTTAAG…5’ 5’…G -OHP-AATTC…3’ 3’…CTTAA -PHO-G…5’ • Note that the above enzyme is EcoRI, the first restriction endonuclease characterized
Most R.E. RecognitionSequences are Palindromes G^AATT-C C-TTAA^G G^GATC-C C-CTAG^G Nodeba Bob Abedon A^GATC-C T-CTAG^G GC^GGCC-GC CG-CCGG^CG
DNA Ligase Upon ligation we now have recombinant DNA
Note that plasmid is vector that carries DNA into recipient cells via transformation Transformation Other vectors include viruses (transduction) as well as otherwise inert projectiles
One example of this might be done… is essentially cloning into animals Gene Therapy
Recombinant Animals Alternatively, DNA can be cloned directly into the germ line
Many plants are easily cloned from individual body cells Cloning into Plants
Cloning allows the amplification of genotype (DNA) as well as phenotype (proteins) Polymerase Chain Reaction If all you really need is the DNA, then PCR is an easy way to amplify DNA without cloning
Rather than making cDNA, one can clone directly into eukaryotic cells, which addresses concerns that bacteria do not modify proteins post-translationally to the extent that eukaryotes do Complementary (c)DNA
2-D Protein Electrophoresis Visualized proteins
Restriction Fragment Analysis No need to know details of what gene was studied or specific restriction enzyme used
Online Tools • Buying primers (custom oligos): http://www.qiagen.com/ • 2x2 sequence comparisons: http://www2.igh.cnrs.fr/bin/align-guess.cgi • n x n sequence comparisons: http://www.genebee.msu.su/services/malign_reduced.html • Sequence manipulation: http://arbl.cvmbs.colostate.edu/molkit/manip/index.html • Sequence translation: http://arbl.cvmbs.colostate.edu/molkit/translate/index.html • Restriction Enzymes: http://rebase.neb.com/rebase/rebase.html • Restriction sites: http://www.ccsi.com/firstmarket/cutter/cut2.html • Sequence searches: http://www.ncbi.nlm.nih.gov/blast/ • Other tools: http://molbiol-tools.ca/