MBMB 451A Section One - Fall 2007 

Molecular Cloning

A.  Cloning Vectors
	1.  Plasmids
		a.  Origin of replication, determines # of copies per cell
		b.  Marker genes; examples are ampicillin and tetracycline resistance genes
		c.  Unique restriction enzyme cut sites:  polylinker / MCS
		d.  Size limitation is ~15,000 bp
2.  Choice of how to get DNA into the cells
		a. chemical transformation
		b. electroporation - especially good for large DNAs
3.  Expression Vectors
		a.  strong promoter for transcription
		b.  ribosome binding site
		c.  transcription terminator
		d. some way of controlling gene expression - inducible
		e.  different tags or fusion proteins
			i.  fluorescent tag - GFP
			ii.  epitope tag
			iii.  metal chelator
4.  Shuttle vectors
		a.  contains origins of replication from two different organisms
		b.  can be replicated in both
		c.  frequently used to shuttle plasmids from bacteria to yeast
5.  Cosmids - a hybrid of plasmid and lambda bacteriophage
6.  Bacteriophages - example of lambda
		a.  1/3 of genome is not essential
		b.  DNA is packaged into phage particles
		c.  can only fit 40-53 Kb of DNA in to phage particles
		d.  In vitro packaging systems
		e.  Highly efficient for transforming DNA
		f.  size limitation is up to 23 Kb of DNA
7.  Bacterial Artificial Chromosomes (BACs)
		a.  100 to 300 Kb in size
		b.  Have selectable markers
		c.  Stable origin of replication
		d.  Used with electroporation
8.  Yeast Artificial Chromosomes (YACs)
		a. ARS or origin of replication
		b.  Selectable markers
		c.  CEN or centromere sequence for proper segregation
		d.  Telomere sequences–
		e.  Suitable for very large DNAs


DNA Libraries
1. Collection of DNA clones
2.  Source for gene discovery
3.  Largest example is a genomic library
4.  Other subsets would be such things as 
		a.  cDNA libraries
5.  Can scan these libraries by DNA hybridization
 
Nucleic Acids  |  Methods-Nucleic Acids: Part A and Part B  | DNA Supercoiling  | Transcription: Part A and Part B  |  Nucleosome | Higher Ordered Structure  |  Chromatin Transcription  |  Regulation of Transcription  |  Course Material

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Last updated on August 14, 2007.

 
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