BCHM 3050 1st Edition Lecture 17Outline of Last Lecture I. Information ProcessingII. Cruciform DNAIII. EcoR1 Restriction SiteIV. DNA Gel ElectrophoresisV. Sanger SequencingOutline of Current Lecture I. How Sanger Sequencing WorksII. Southern BlottingCurrent LectureI. How Sanger Sequencing Worksa. Based on sticky ends, you can introduce foreign DNA into a piece of existing DNAb. DNA strands are complementary to each otherc. Phosphodiester bonds exist between nucleotides (need an OH group at the 3’ end)d. Sanger generated di-deoxynucleotides - ddGtP, ddCTP, ddTTP, ddATPe. He replaced the OH with just an H, which cannot form a phosphodiester bond à cannot extended the DNA chainf. Has 4 tubes: - g. #1 adds 1 strand of DNA + dATP/CTP/GTP/TTP+ ddATPh. #2 adds 1 strand of DNA + dATP/CTP/GTP/TTP+ ddGTPi. #3 adds 1 strand of DNA + dATP/CTP/GTP/TTP+ ddCTPThese 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.j. #4 adds 1 strand of DNA + dATP/CTP/GTP/TTP+ ddTTPk. These di-deoxynucleotides prevent the DNA chains from being able to extendl. Radioactively labeled the phosphate groups of the “dd” versions of the nucleotidesm. He is trying to recreate the bottom strand of the original DNAn. Read 5’ to 3’, bottom to topo. Color code nucleotides – by measuring the intensity of the color, can determine the sequenceII. Southern Blottinga. Much less used procedureb. Uses restriction enzymes to cut up large strand of DNA into tiny piecesc. Two pieces of DNA that he is interested in are darkly shadedd. Runs all pieces of DNA in agrose gele. Figures out where DNA of interest is by sending a probe which is the actually sequence of DNA, labels it, and adds it to the DNA gelf. Can also put filter paper on top of gel and filter paper will absorb all pieces of DNA through adhesiong. Send radioactive probe and whereever the DNA of interest is, the probe will light uph. Southern Blotting isnt very helped but can tell you if your DNA of interest is present, doesn’t tell you exactly where to find
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