Dave Gray's Build-A-Gene Class Notes - Session 5
Session 5 included two activities:
- We screened the DNA in our bacteria for size
- Although we didn't actually sequence the gene, we discussed the process for doing so.
To size screen our gene, we performed "Colony Screening PCR". First, we amplified the gene portion of our vector. This involved taking 8 samples of the bacteria using a toothpick and swirling each in its own vial contiaining 50μL of water. We then adding two primers (one for each end of the gene) and PCR master mix (nucleotides, Taq and buffer). These were then run through the PCR machine. The primers select the two ends of the emGFP gene so that just that portion of the vector gets amplified. The PCR machine starts out at a temperature of 95°C for 6 minutes to burst the bacteria. Then, the other items added can access the genetic material and the amplification can proceed.
After the amplification was complete, we ran the samples through gel electrophoresis to identify samples that appeared to have genes of the correct length.
Note: When taking sample bacterial colonies, we looked for locations where a colony was round in shape. That suggests that the colony grew from a single bacteria. Taking the sample just requires a gentle swab with the toothpick, not gouging the agar. Also, when working with live bacteria, we were advised to keep the vials capped to prevent airborne bacteria and other organisms from polluting our product.
Because this was our last session, we did not have an opportunity to send the gene out for sequencing. However, we discussed the process and available tools. The technique is called "Sanger", "Chain Termination" or "Cycle" sequencing. There is an alternate process called "next generation sequencing" used for larger scale work.
This sequencing technique starts with primers - typically the same as those used in colony screening PCR. These are mixed with a large set of deoxynucleotides, all with a 5' phosphate group (PO4) on one end and a 3' hydroxyl (OH) on the other. Then a smaller volume is added of dideoxynucleotides with a 5' phosphate group but no 3' hydroxyl (OH). These also have an attached phosphorescent protein. The dideoxynucleotides will stop DNA synthesis where they are inserted because of lacking a 3' end. By working with a large number of copies, samples with each length are produced. We then use a process similar to gel electrophoresis to sort them by size. By tracking the sequence of phosphorescent colors, we can determine the nucleotide sequence.
This works for up to about 400 nucleotides from each end. Then the nucleotides become long enough that the tend to "bunch up" with various lengths traveling at a similar pace. However, we can do the test from both ends of the gene and have some overlap to validate our results. So our 750 nucleotide emGFP gene can successfully be sequenced in this way.
We looked at some websites that help work with sequencing results. The first shows a color-coded electropherogram which would generally accompany the resulting sequence. It represents the data obtained from sequencing detector, with the height of each peak representing the strength of the signal. We can therefore see the quality of the sequencing data that was obtained as well as investigate any ambiguities in the sequence. A sample electropherogram is here.
When we receive our results, we need to check the letter sequence against what we planned. Rather than do that manually, we can use a site called Clustal W. To include the reverse sequence that started from the end of the gene and worked back, we have to reverse the order of the letters. A site called Sequence Manipulation Suite can help with that.