MBMB 451A Section One - Fall 2007

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Methods for Working with DNA and RNA  (Part B)

6.  DNA sequencing - two methods
	A.  Sanger or the dideoxy method (enzymatic)  
		i.  use of dideoxynucleotides as chain terminators
  		    will have four separate reactions for each chain terminator figure 7-14 and 7-13 
		ii.  Klenow fragment of DNA polymerase
		iii.  single-stranded DNA template annealed to an oligonucleotide primer  
		iv.  alpha P-32 dATP typically used to internally labeled synthesized DNA figure 7-15
	B.  Automated DNA sequencing
		key is the fluorescent labeling of DNA using fluoresently tagged dideoxynucleotides figure 7-16

7.  DNase I footprinting and Interference Assay
	A. radiolabel one unique 5' end of the DNA using T4 polynucleotide kinase and alpha-P32 ATP.
	B.  after binding protein to DNA, add in DNaseI (an endonuclease)  to cut the regions of DNA not bound by protein
	C.  It is key to cut the DNA only once per DNA molecule figure 

	Will discuss Interference Assay in lecture

8.  cDNA   figure 
	A. an oligo dT primer is annealed to the poly A portion of the mRNA template
	B.  the enzyme reverse transcriptase catalyzes the synthesis of DNA from an RNA template
	C.  reverse transcriptase backs around to place a "hook" at the end - DNA-DNA primer
	D.  RNA is removed by alkaline hydrolysis 
	E.  DNA synthesis is completed with DNA polymerase
	F.  this process is used in molecular cloning see next section

9.  PCR or polymerase chain reaction figure 5-54 
	A.  An effective method for in vitro amplification of a particular DNA sequencing
	     as much as a million times amplification with 25 cycles
	B.  relies on using a thermostable version of DNA polymerase
	     example is Taq DNA polymerase
	C.  can be used for 
		molecular cloning (also RT-PCR, combine PCR with synthesis of cDNA)
		site-directed mutagenesis
		rapid identification of clone
		rapid DNA sequencing  to name just a few
	D.  Extreme care needs to be taken to prevent contamination
	      even dust can be significant

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Nucleic Acids  |  Methods-Nucleic Acids: Part A  |  DNA Supercoiling  |  Nucleosome  | Transcription: Part A and Part B  |  Higher Ordered Structure  |  Chromatin Transcription  |  Regulation of Transcription  |  Course Material

 

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Last updated on August 14, 2007.