9-notes - 580.221 BME Molecules and Cells L09 Recombinant...

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580.221 BME Molecules and Cells L09 Recombinant DNA p 1 Lecture Nine – Recombinant DNA techniques Outline: Tools to make & manipulate DNA Tools to analyze DNA Cloning, PCR, Sequencing Recombinant DNA Technologies Recombinant DNA Technologies are 1) tools to make and manipulate DNA 2) tools to analyze DNA Questions that can be answered with these tools include: I have a protein of interest, what is the gene? [difficult to sequence protein, try gene] what is the family? [based on sequence homology ] What is the protein’s function? I have an interesting gene, what are the mutations in the gene? when is the gene expressed? what controls its expression? - can introduce mutations in the DNA and see what happens to the protein. Tools to make and manipulate DNA De Novo Synthesis : stepwise construction of DNA molecules starting from a solid support. Use nucleotides that have a protective group (DMT-O) which prevent polymerization from proceeding past a single step. Then remove the excerss nucleotides and modify the group for the next polymerization reaction. This method is limited to around 100 bp, because the yield at each step is less than 100%. Cut and Paste Methods : remove a fragment from one DNA strand and add the fragment to another DNA strand. Restriction enzymes are useful for this purpose (See below.) Amplification : Using a small quality of DNA to create more DNA of the same sequence. Cloning and PCR are two methods of amplification (See below.) Tools to analyze DNA Sequencing : Using techniques such as the Sanger Method (see below) to determine what base pairs a piece of DNA contains, and in what order they appear. Summary of Sanger (dideoxy) Method : The current way to sequence DNA. Single stranded DNA (to be sequenced) is added to a solution with A,C,T, and G. A small percentage of these bases are dideoxy, meaning that when they are incorporated, additional bases will not add to the molecule. Polymerases included in the mixture build the second strand until a dideoxynucleotide is added. The resulting DNA strands vary in weight according to how many nucleotides have been added, causing them to cluster in different bands when run out on a gel. Although radiolabelling was previously used, modern sequencing relies on fluorescence. Each of the 4 bases emits at a different wavelength, allowing them to be easily recognized on the gel. Recall
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580.221 BME Molecules and Cells L09 Recombinant DNA p 2 that only one fluorescent dideoxynucleotide is incorporated into each strand. The sequence of the DNA can be read directly from the fluorescing gel. (see lecture 6 for more) Limitations of DNA sequencing DNA strands longer than 200 base pairs are very difficult to sequence. This occurs because larger molecules run very close together on gels, making it difficult to separate large sequences that differ by only one base pair. Circumventing Sequencing Limitations
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9-notes - 580.221 BME Molecules and Cells L09 Recombinant...

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