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Trends in Biotechnology
Chapter 3a – Tools forRecombinant DNA
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Chapter 3 - outline
II.Cutting and Joining DNA
III.Separating Restriction Fragments and VisualizingDNA
IV.DNA Cloning
V. Cloning Vectors
A. Bacterial Vectors
1. Plasmids
2. Bacteriophage
3. Cosmids
B. Vectors for Other Organisms
1. Yeast Artificial Chromosomes (YACs)
2. Bacterial Artificial Chromosomes (BACs)
3. Plant Cloning Vectors
4. Mammalian Cell Vectors
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VI. Cell Transformation
VII. Constructing and Screening a DNALibrary
A. Genomic Library
B. cDNA Library
C. Screening Libraries
D. Expression Libraries
VIII. Reporter Genes
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IX.Southern Blot Hybridization
X.Northern Blot Hybridization
XI.Polymerase Chain Reaction
XII.DNA Sequencing
XIII. Protein Methods
A. Protein Gel Electrophoresis
B. Protein Engineering
C. Protein Sequencing
XIV. DNA Microarray Technology
A. Biotech Revolution: RNA InterferenceTechnology: Gene Silencing
XV. Applications of Recombinant DNATechnology
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Learning Objectives
1. Know how restriction endonucleaseswork to cut DNA, and why they areimportant in biotechnology. Compare bluntends with sticky ends.
2. Know how electrophoresis separatespieces of DNA.
3. List and know the steps of DNA cloning.
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4. Know how vectors are used totransform bacteria, and how to select forsuccessfully transformed bacteria.Compare the how different vectors cancarry different sizes of DNA into thebacteria.
5. List the types of vectors that can beused to transform yeast, mammalian cellsand plants, and why they are effective inthose organisms.
6. List the methods of transformation ofcells.
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7. Compare genomic libraries, cDNAlibraries, and expression libraries. How arethey constructed? What are the librarieslooking for? How they are screened?
8. List the various types of reporter genesused in research.
9. Compare Northern and Southern blothybridization. How are they constructed?What is each type of hybridization lookingfor?
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10. Know the function of PCR, the steps ofPCR, and what researchers do with PCR.
11. Compare the two methods of DNAsequencing: the chemical method and theSanger method, and know which methodis more widely used. How doesautomation change DNA sequencing?
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12. List and define the various methods ofanalyzing proteins. Are any of thesemethods similar to DNA methods?
13. Know the types of microarrays, and howDNA and protein microarrays work.
14. List the applications of recombinantDNA technology.
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Fig. 3.1 Cutting double-stranded DNA.
(a) The enzyme DNase cuts DNA atrandom sites.
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Fig. 3.1 (b) Restriciton enzymes cutDNA at specific sites.
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http://www.nature.com/scitable/resource?action=showFullImageForTopic&imgSrc=44956/pierce_18_2_FULL.jpg
Figure 3 a:Restrictionenzymesmake double-stranded cutsin the sugar-phosphatebackbone ofDNA
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Fig. 3.1 (c) A Specific restrictionenzyme cuts each DNA molecular atthe same sequence site.
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Fig. 3.2 The ligation of two different pieces of DNA.
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Figure 3 b:Restrictionenzymesproducingcohesive, orsticky, ends.
These ends canbe joinedagain.
http://www.nature.com/scitable/resource?action=showFullImageForTopic&imgSrc=44956/pierce_18_2_FULL.jpg
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Video: restriction enzymes bio37
(13 restriction)
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Fig. 3.3 Agarose gel electrophoresis is used to separateDNA (and RNA) molecules according to size.
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Electrophoresis:
(20 Electrophoresis - gelelectrophoresis.exe)
Together, restriction enzymes and gelelectrophoresis can give a lot ofinformation.
(20 Electrophoresis - rest enzy and electro.exe)