Dave Gray's Build-A-Gene Class Notes - Session 2: Difference between revisions
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In session 2, we assembled the emGFP coding sequence from smaller segments. We also used gel electrophoresis to check the results of our Session 1 PCR. | In session 2, we assembled the emGFP coding sequence from smaller segments. We also used gel electrophoresis to check the results of our Session 1 PCR. | ||
The emGFP coding sequence is about 750 nucleotides long. We ordered this in 60 nucleotide sets - about 20 pieces. That means that what we ordered was about 1200 nucleotides. The reason this is greater than the 750 nucleotide target is that the pieces are designed to overlap by about 20 nucleotides so that the segments will line up correctly. Each strand includes a 3' OH for the polymerase to attach to so that it can fill in the gaps. We use PCA (Polymerase Chain Assembly) to accomplish this. | The emGFP coding sequence is about 750 nucleotides long. We ordered this in 60 nucleotide sets - about 20 pieces. That means that what we ordered was about 1200 nucleotides. The reason this is greater than the 750 nucleotide target is that the pieces are designed to overlap by about 20 nucleotides so that the segments will line up correctly. Each strand includes a 3' OH for the polymerase to attach to so that it can fill in the gaps. We use PCA (Polymerase Chain Assembly) to accomplish this. Controlling the temperature helps ensure that segments with flaws in the overlapping sections do not successfully anneal. | ||
The reason for ordering shorter segments is that it helps ensure that we get some error free emGFP coding sequences. The error rate for chemically synthesizing DNA is .995 (5/1000). So in a 1000 nucleotide segment, on average, we are likely to get 5 errors. Or for 750 nucleotides (for emGFP), the number would be closer to 4. | The reason for ordering shorter segments is that it helps ensure that we get some error free emGFP coding sequences. The error rate for chemically synthesizing DNA is .995 (5/1000). So in a 1000 nucleotide segment, on average, we are likely to get 5 errors. Or for 750 nucleotides (for emGFP), the number would be closer to 4. |
Revision as of 17:37, 7 August 2013
In session 2, we assembled the emGFP coding sequence from smaller segments. We also used gel electrophoresis to check the results of our Session 1 PCR.
The emGFP coding sequence is about 750 nucleotides long. We ordered this in 60 nucleotide sets - about 20 pieces. That means that what we ordered was about 1200 nucleotides. The reason this is greater than the 750 nucleotide target is that the pieces are designed to overlap by about 20 nucleotides so that the segments will line up correctly. Each strand includes a 3' OH for the polymerase to attach to so that it can fill in the gaps. We use PCA (Polymerase Chain Assembly) to accomplish this. Controlling the temperature helps ensure that segments with flaws in the overlapping sections do not successfully anneal.
The reason for ordering shorter segments is that it helps ensure that we get some error free emGFP coding sequences. The error rate for chemically synthesizing DNA is .995 (5/1000). So in a 1000 nucleotide segment, on average, we are likely to get 5 errors. Or for 750 nucleotides (for emGFP), the number would be closer to 4.
Another way of calculating this is use the probability of a correct outcome for each nucleotide added - .995 or 99.5%. To calculate the likelihood of any series of nucleotides being correct, we need to raise .995 to the power of the number of nucleotides. So for a 750 nucleotide emGFP sequence, we would calculate (.995)750 = .027 or 2.7% of the resulting strands being correct. By reducing the length to 60, we get (.995)60 = .74 or 74% of the segments are good. We will amplify these to produce many copies and then join them. As a result, we should have many perfect sequences - along with many more imperfect. So we will have to weed out the ones we want.
Chemical synthesis of DNA uses phosphoramidite nucleotides, a modified precursor to DNA. Later we end up with real DNA.
We will be using herculase rather than taq polymerase because of it's better error rate. (My notes say 1 in 8 vs 1 in 12, but unclear what this means.)