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Virginia Tech BCHM 4116 - Restriction Enzymes and the manipulation of DNA

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BCHM 4116 1st Edition Lecture 6 Outline of Last Lecture I. Genome size vs complexity II. DiversityIII. Evolution Outline of Current Lecture IV. Basic manipulation of DNA V. Restriction Endonucleases and hydrolysis of nucleic acidsVI. Type II Restriction Endonucleases VII. Related Issues VIII. Ligation of Restriction Fragments Current LectureBasic Manipulations of DNAIsolation of DNAThe 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 DNA3. 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 AcidsIn order to do restriction enzymes, there are two ways to break DNA up. One is physical methodwhere 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 Acidsa. This can be done in one of two ways. i. Acid or base hydrolysis ii. Nucleases (enzymatic hydrolysis) 2. Enzymatic Hydrolysis of Nucleic Acidsa. DNase, RNase, and non-specific nucleases (works on both RNA and DNA)b. Exo vs Endonucleases i. Endo: cuts in the middle of the strandii. 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 DNAc. 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, ExamplesThese 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 FragmentsLigation 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


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