BICH/GENE 431 KNOWLEDGE OBJECTIVES
Chapter 21 – Methods
- palindromic recognition sequences
- overhanging ends (5’ or 3’) vs. blunt ends after cutting
- probability of occurrence of cutting depending on length of rec. seq.
- role of methylation
Electrophoresis of nucleic acids
- agarose vs. polyacrylamide gels
- charge on nucleic acid and direction of electrophoresis
Blots – general idea; Southern blot (DNA); northern blot (RNA)
Recombinant DNA; DNA cloning – what do they mean?
Types of cloning vectors used in E. coli and rationale for using various types
Basic features of a plasmid
DNA libraries – genomic vs. cDNA; what is difference?
How is cDNA made?
What is difference between a selection and a screen?
How to screen a library?
- inventor Kary Mullis
- some uses
- describe steps and how it results in exponential amplification of DNA
Describe site-directed mutagenesis of DNA – Quickchange method
Dideoxy sequencing method
- inventor Fred Sanger
- steps; how does it work?
- role of dideoxy nucleoside triphosphates as chain terminators
Next generation DNA sequencing
- names of four different technologies
- general idea in comparision to dideoxy sequencing
- understand basics of 454 (pyro sequencing)
- understand basics of Solexa sequencing
- what are benefits and limitations?
Basic strategy behind genome sequencing
- contigs, scaffolds, “personal genomes”
- understand basic technique and general usage
- SDS-PAGE: what is SDS and what does it do?
- Role of beta-mercaptoethanol or dithiothreitol to reduce disulfide bonds
- Immunoblots (western blots) – what are they and basic technique
What is the proteome? What are some goals of proteomics research?
General ideas behind protein identification and sequencing by mass spectrometry
Electrophoretic mobility shift assay (EMSA) – understand basic technique and use
Footprinting – understand basic technique and use
Compare some aspects of EMSA and footprinting
Chromatin Immunoprecipitation (ChIP) – understand basic technique and use
SELEX or in vitro selection – understand basic technique and use