chapter 28 manipulating dna. chapter objectives know how the techniques of molecular biology work...
TRANSCRIPT
Chapter 28
Manipulating DNA
Chapter Objectives
• Know how the techniques of molecular biology work
• Understand how to use the tools of molecular biology
• Put it all together with a molecular biology project (we will work on this in class after we know the tools and techniques)
Techniques
• Restriction Enzymes• Electrophoresis• Hybridization probes• DNA sequencing– Chain terminator– Automated– Emulsion
• PCR
Restriction Enzymes
• https://www.neb.com/products/restriction-endonucleases
• App: Search NEB tools in either play store or App store
• Remember overhangs, this will be important
Electrophoresis
Polyacrylamide
Agarose
Hybridization Probes
• Northern Blot – RNA detection• Southern Blot – DNA detection• Western Blot – Protein detection
DNA Sequencing
• Chemical Sequencing
• Chain Terminator (dideoxy nucleotides)– Automated
(color)• Emulsion
PCR
95denature
55?annealing
72elongate
repeat
Tools
• RT-PCR• Microarray• Cloning– Plasmids– Bacteriaphage– Cosmids– BAC/YAC
• Site-directed mutagenesis• Knockout mice
Real-time (kind of) PCR(qPCR) (sort of)
• Can quantify the exact about of mRNA using PCR
• Combine dsDNA binding dye with PCR
Microarrays
Cloning
• Plasmid – small, circular DNA – can hold about 10 kb
• Bacteriophage – larger, circular DNA – can hold about 20 kb
• Cosmid – combination of a plasmid and phage, can hold 40-50 kb
• BAC/YAC – bacterial artificial chromosome/yeast AC – can hold up to 500 kb
Site Directed Mutagenesis
• Change DNA in plasmid, etc. to make a mutation in protein (substitution/deletion)
• Can change any residue to any other residue– Implications?
Mice
• Knockout– Deletion of gene
• Knockin– Insertion of gene
• Genetic background– models
Active Learning
• Projects– Download serial cloner• http://serialbasics.free.fr/Serial_Cloner.html
– Build plasmids (learn about functions)– Create (on paper) a mutant protein• Create an experiment to study your protein
– Learn about creating knockout mice.