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- 1. Open EndedTo familiarize students with the science and technology used to identify types of DNA based on their DNA sequence.
- 2. Open EndedPrepare a sample and isolate the whole DNA, make copies of it, sequence the DNA, analyze and identify
- 3. Open EndedThe piece of DNA used for identifying bacteria; The identification relies on matching the sequence from your sample against a database of all known 16S rDNA sequences
- 4. Open Endedto identify a bacterial sample received from a clinician.
- 5. Open Endeddissolving the cell wall with a digestive buffer (in the white-capped bottle) available as a commercial kit
- 6. Open Ended
- 7. Open EndedThe buffer contains proteolytic enzymes that eat the cell wall.
- 8. Open EndedSince we will be using other enzymes in the next step, we need to get rid of the proteolytic enzymes.
- 9. Open EndedDNA is lighter than the cellular debris floating around from the step that destroyed the cellular wall. The centrifuge allows us to safely remove the DNA from the microcentrifuge tube.
- 10. Open Endeda solid deposit of cellular debris
- 11. Open Endedthe liquid
- 12. Open Endedthe DNA is contained in the supernatant
- 13. Open EndedPolymerase Chain Reactant; makes copies of DNA
- 14. Open Ended
- 15. Open Endedwater, a buffer to maintain pH, nitrogenous bases
- 16. Open Endedsmall pieces of DNA that bind to specific sequences
- 17. Open EndedThe hotness is then elevated once again to 72 degrees Celsius.
- 18. Open EndedExtremely similar among different species
- 19. Open Endeduniversal primers bind to them so that they can be used to copy DNA from a variety species of bacteria.
- 20. Open Endedgenes that differ among different species; used for identification.
- 21. Open Ended
- 22. Open EndedDNA
- 23. Open EndedThe only thing present in the positive control tube is the positive control DNA while the negative control tube contains only sterile deionized water
- 24. Open Ended
- 25. Open Ended
- 26. Open Ended25
- 27. Open Ended1,000,000
- 28. Open Ended1500 bp long
- 29. Open Ended
- 30. Open EndedOne lane should be the negative control only containing water, there shouldn't be anything in the lane unless the water is contaminated. The middle lane should be the positive control containing the PCR known product, which is to make sure that the PCR itself worked. The last lane is for our unknown sample.
- 31. Open Endedshould now have many pieces of 1,500bp-long 16S rDNA, with a very small amount of longer DNA strands (which are containments)
- 32. Open Ended
- 33. Open EndedThey both contain a "sequencing brew" which consist of buffers, primers, DNA polymerase, nucleotides and fluorescence-tagged terminators in suitable proportions. The PCR product from the previous step is added to each tube and another PCR is run. The aim is not produce identical copies of DNA but many copies of variable length. Each DNA strand binds the primer at one end and will have a fluorescence-tagged terminator at the other end.
- 34. Open Endedto produce many sequences of varying lengths using primers to copy sections
- 35. Open EndedThe are available from a commercial source
- 36. Open Endeda mix of DNA of variable lengths
- 37. Open Endedseparates molecules based on differences in size and charge
- 38. Open EndedDNA molecules are negatively charged and they start to move through the tube towards the positively charged syringe end, with the smaller pieces moving faster than the larger ones.
- 39. Open EndedIt excites the fluorescent markers, and optical detectors detect the color of the fluorescence by reading the sequence of nucleotides based on their fluorescence as the DNA pieces pass through the laser beam, sequences of the DNA can be reconstructed
- 40. Open EndedSequence of the DNA can be reconstructed
- 41. Open Endedtwo sequences of one similarity
- 42. Open EndedBasic Local Alignment Search Tool; matches DNA sequences to determine the bacteria species found
- 43. Open EndedThe number of positions that differ in the nucleotide sequence is proportional to the time elapsed since the two species formed their own lines of descent from a common predecessor
- 44. Open Ended"Score" (bits)" means an indication of how good the alignment is; the higher the score, the better the alignment is
- 45. Open EndedIt refers to the expected number of random hits for a given alignment score. It represents a more reliable match
- 46. Open EndedBartonella sp
- 47. Open EndedIt is a genus of Gram-negative bacteria. Only genus in the family of Bartonellaceae. It can infect healthy people, but they are considered important as opportunistic pathogens
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