BCHM 4116 1st Edition Lecture 6 Outline of Last Lecture I Genome size vs complexity II Diversity III Evolution Outline of Current Lecture IV Basic manipulation of DNA V Restriction Endonucleases and hydrolysis of nucleic acids VI Type II Restriction Endonucleases VII Related Issues VIII Ligation of Restriction Fragments Current Lecture Basic Manipulations of DNA Isolation of DNA The cell is lysed to remove cell debris 1 To remove protein you use phenol protease and ion exchange column a Protease doesn t affect DNA or RNA only enzymes 2 To remove RNA you use RNase which doesn t affect DNA 3 DNA is precipitated by using ethanol DNA fragments then have to be separated The fragments are placed In a gel matrix at the negative charge end and due to charge separation the DNA fragments travel to the other end positive end The smallest fragments elute first and the largest fragments elute last At the end it is gel electrophoresis that used to separate DNA fragments These notes represent a detailed interpretation of the professor s lecture GradeBuddy is best used as a supplement to your own notes not as a substitute Restriction Endonucleases and Hydrolysis of Nucleic Acids In order to do restriction enzymes there are two ways to break DNA up One is physical method where you traumatize the DNA and it breaks An example of this is putting it through a very small needle to shred it Methods like this are good to make random fragments Other methods include enzymes that break phosphodiesterase bond at certain sections of the DNA Depending on how the DNA breaks you could end up with a 3 hydroxyl and a 5 phosphate or a 3 phosphate or a 5 hydroxyl 1 Hydrolysis of Nucleic Acids a This can be done in one of two ways i Acid or base hydrolysis ii Nucleases enzymatic hydrolysis 2 Enzymatic Hydrolysis of Nucleic Acids a DNase RNase and non specific nucleases works on both RNA and DNA b Exo vs Endonucleases i Endo cuts in the middle of the strand ii Exo cuts at end of the strand 3 Restriction Endonucleases a Type I and III require ATP modifiy DNA by methylation b Type II Enzymes require Mg2 no modification of DNA c These cleave DNA at or near palindromic recognition sequence 4 8 bp In the first paper discussion there are sequences of introns exons and palindromic sequences Type II Restriction Endonucleases Examples These cut at specific sites and can result in the formation of blunt ends or sticky ends Blunt ends are where there is no overhang from the cut Stick ends can result in a 5 overhang or a 3 overhang Compatible ends are when two enzymes may produce the same sticky ends at different recognition sites Methylation ability of different enzymes to cut methylated DNA varies Ligation of Restriction Fragments Ligation occurs when there are complementary stick ends A 3 hydroxyl and 5 phosphate will bind to one another via a ligase and ATP and form a whole fragment
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