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.