LECTURE 7&8-You say you want a revolution DNA analysis methods

LECTURE 7&8-You say you want a revolution DNA analysis methods

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DNA methods summary 1. Restriction enzymes  cut at specific DNA sites.  (N) 2. Vectors  allow genes to be “cloned” and proteins  “expressed”.  (N) 3. Gel electrophoresis  separates DNA on the basis of  size. 1. DNAs can be  synthesized  (up to  ~ 100 bases  commercially).  (N) 1. PCR  amplifies any target DNA sequence.  (N) 1. Genes and genomes can be  sequenced  by chain  termination.  (N) 2. Oligonucleotides can be used to change bases by  site-directed mutagenesis ”.  (N) 1. “Southern” blotting  detects sequences by  hybridization. 1. Genes can be  knocked out   (deleted)  or  replaced  in  prokaryotes and eukaryotes.  (N) 1. Microarrays  detect gene expression patterns over the  genome. 
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Restriction enzymes cut DNA at specific sites
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Restriction enzymes cut DNA at specific sites Palindrome
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Restriction enzymes cut DNA at specific sites Palindrome note, I dissent. A fast never prevents a fatness. I diet on
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Restriction enzymes cut DNA at specific sites  types of ends: 5’ overhang, blunt and 3’ overhang ognate methyl transferases protect host genome from digesti Restriction-modification systems degrade “foreign” DNA.
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Average frequency of restriction sites in “random” DNA sequences e average occurrence of each sequence = 1/4 n , ere n = the site length and all bases are equally represent Site size 4 6 8 Average frequency  1/256 1/4,096 1/65,536 (1/4 x 1/4 x 1/4 x 1/4)
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Lots of different recognition sites known Core four bases Flanking bases None A----T C----G G----C T----A
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A simple cloning procedure 1. Cut “insert” and  “vector” DNA with a  restriction enzyme 1. Mix and join ends  with DNA ligase. The  ends should match  for efficient  ligation.
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Cloning without DNA ligase 1. Prepare open vector and  insert with the same long  “sticky” ends     + Pol I Klenow fragment +  dATP 1. Mix and let the ends  anneal. 1. Transform the nicked 
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LECTURE 7&8-You say you want a revolution DNA analysis methods

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