MBMB 451A Section One - Fall 2007

Methods for Working with DNA and RNA (Part A)

1.  Gel electrophoresis
	A.  Materials: agarose (large DNAs) vs. acrylamide (high resolution, DNA sequencing)
	B.  Separated by its sieving property and charge:  both are proportional to size of DNA
	C.  Large DNAs are resolved by pulse field gel electrophoresis
		1 min in forward direction and 15 seconds in reverse direction figure 
	D.  Visualize DNA by staining with such things as ethidium bromide
		intercalates into DNA and flouresces, detection limit is near 10-20 ng


2.  Enzymes
	A.  Restriction Endonucleases - table 
		i.  originate from a host-specific modification
			a. host restriction endonuclease
			b. host modification methylase
		ii.  three types of endonucleases
			a.  type I and III are single polypeptides with both endonuclease & methylase activity
			b. type I cuts at least 1000 bps away from recognition site and type III within ~24-26 bps
			c. type II has separate endonuclease and methylase activity, and cuts at the specific recognition site
			d.  sticky ends vs blunt ends - figure 5-37 
	B.  Restriction Mapping and RFLP mapping
		i.  restriction cut sites are physical reference points on a DNA molecule - figure 5-39 and 5-40		
		ii. Restriction Fragment Length Polymorphisms: RFLP - figure 5-41
			a.  DNA fingerprinting in criminal cases
			b.  genetic screening for particular diseases see Southern blotting
	C.  Exonuclease
		i.  3' exonuclease
		ii.  5' exonuclease
	D.  Others - table

3.  Southern Blotting figure 5-50
	Purpose: is to identify the presence of a particular DNA (RNA) sequence in you sample.
			agarose gel - electrophoresis
					I
			treat with sodium hydroxide (denature)
					I
			transfer to nitrocellulose membrane
			(have nylon or reinforced membrane)
					I
			fix on to membrane - by baking or UV crosslinking
					I
			annealing step:
				modes of detection:  primarily radioactivity or chemilliminescence

Type of Blot	Identify	Probe or manner of detection
Southern	DNA	using DNA or oligonucleotide probe
Northern	RNA	using DNA or oligonucleotide probe
Western	protein	using specific antibodies, electrophoretic transfer membrane, block membrane with BSA or milk protein
Far Western	protein	for detecting one protein interacting with another protein
South Western	protein	using a radiolabeled DNA probe

	              example

4. DNA Micro Chip Arrays- Genomic wide analysis (see Nature Biotechnology 16, 27-31 1998) figure 
 	A.  A thousand different DNAs are immobilized onto a glass slide using the same technology 
                  used by computer chip manufacturers
	B.  Fluoresecently tag total cellular mRNA from wild type cells and anneal to immobilized DNA
	C.  Quantitate the total amount of each individual mRNA by the amount of fluorescence 
	     at any given position in the slide, sensitivity ranges from 0.1-100 mRNA/cell 
	D. Can determine the effect of one gene product on the genome wide expression of mRNA 
	    by isolating mRNA from deletion strain missing that gene or from a strain with a temperature sensitive strain.
	    See example below

                                                                                       figure courtesy:
                                                                          Cell, Vol. 95, 717Ð728, November, 1998, Copyright © 1998 by Cell Press
                                                                          Dissecting the Regulatory Circuitry of a Eukaryotic Genome
                                                                          Frank C. P. Holstege1, Ezra G. Jennings, John J. Wyrick, Tong Ihn Lee, Christoph J. Hengartner,
                                                                          Michael R. Green, Todd R. Golub, Eric S. Lander, and Richard A. Young

5. Molecular Cloning and Recombinant Protein Production

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Last updated on August 14, 2007.