Molecular Identification of Bacteria: 16S rRNA Sequence PDF

Molecular identification of unknown bacteria using 16S rRNA sequence Related Reading Read pages 456-457, 485-498, 508-509 in Staley’s Microbial Life. Learning outcomes: Apply DNA fingerprinting techniques to identify unknown bacteria THIS EXERCISE IS TO BE PERFORMED BY EACH STUDENT INDIVIDUALLY...

Molecular identification of unknown bacteria using 16S rRNA sequence Related Reading Read pages 456-457, 485-498, 508-509 in Staley’s Microbial Life. Learning outcomes: Apply DNA fingerprinting techniques to identify unknown bacteria THIS EXERCISE IS TO BE PERFORMED BY EACH STUDENT INDIVIDUALLY In this laboratory, you will amplify a region of the 16s rDNA gene from the DNA of your unknown enrichment culture strains using universal eubacterial primers and the Polymerase Chain Reaction (PCR). The DNA will be sequenced by Mc Lab. You will then compare the results obtained with the public databases (e.g. NCBI) and determine the identity of your unknown bacteria. Later, you will compare your biochemical tests results to your molecular identification results. Day 1 DNA Extraction and PCR Materials ​ Most recent streak plate on which you isolated your unknown species ​ 1 sterile 0.2 mL tube ​ 20ul of microLYSIS® solution (one larger aliquot per group) ​ 2 PuRe Taq Ready-To-Go™ PCR Beads reaction tubes ​ 1 sterile eppendorf tube ​ Sterile water (om eppendorf tube) ​ Primers (10 um stocks): 27F (5’-AGAGTTTGATCCTGG-3’) and 1492R (5’-TACCTTGTTACGACTT-3’) ​ P200 and P10 pipettes and tips ​ Thermal cycler ​ Vortex ​ Centrifuge ​ Gloves ​ Ice bucket Procedure Each unknown culture should be handled using aseptic techniques. Be careful not to contaminate anything, especially yourself. DNA extraction a)​ Add 20 ul of microLYSIS® solution a sterile 0.2ml tube with a sterile pipette tip. Then use a pipette tip to sample a colony from one your unknown culture plates and resuspend it in the microLYSIS® solution by gentle mixing. Do not pipet because you do not want to make bubbles  here. Do not forget to label your tubes (unknown number). Do not be extremely greedy or you won’t get clean DNA. b)​ Place your tube in the thermal cycler and wait for your instructor. c)​ Cycling profile for microLYSIS (your instructor programmed it in the thermal cycler): Step 1: 65℃ for 5 mins Step 2: 96℃ for 2 mins Step 3: 65℃ for 4 mins Step 4: 96℃ for 1 min Step 5: 65℃ for 1 min Step 6: 96℃ for 30 secs Step 7: 20℃ hold Note - microLYSIS® solution is a complex solution which releases (does not purify) the DNA from the bacteria cells. After cycling (ca. 15 min), all the microLYSIS® solution / DNA mixture can be used directly in PCR and it can be stored at -20℃ for future use. For the amplification of bacterial DNA, 1-3 ul of the microLYSIS® solution / DNA mixture is usually sufficient. 16S rRNA Gene Amplification (PCR reaction) a) Prepare a master mix in the sterile eppendorf tube. It contains forward primer and reverse primer that are specific for a region of 16S rRNA gene amplification and water in this ratio: 1ul of primer 27F x ____________ reactions = ____________ ul 1ul of primer 1492R x ____________ reactions = ____________ ul 21 ul of sterile water x ____________ reactions = ____________ ul b) Add 23 µL of master mix in each of your PuReTaq Ready-To-Go™ PCR Bead tubes (1 unknown and 1 control). Gently tap the tube to dissolve the bead which contains dNTP, Mg2+, PCR buffer component, and Taq DNA Polymerase for a 25 ul PCR reaction. Keep your tube on ice. c) In that first PuReTaq Ready-To-Go™ PCR Bead tube, add 2 µL of the microLYSIS® /DNA mixture from your unknown. Label your tube and keep on ice. d) In your control PuReTaq Ready-To-Go™ PCR Bead tube, add 2 µL of sterile water in place of DNA for your negative control. Since no DNA is added to this tube, amplification should not occur. This checks for unwanted contamination. e) Tap your PCR tubes gently to mix all the components well. Till now you have every component for a 25 ul PCR reaction: DNA template Primers Mg2+ PCR buffer H2O Taq DNA polymerase  Total volume: 25 ul PCR Cycling: Step 1: 94C for 2 min Step 2: 94C for 45 sec ------ denaturation Step 3: 55C for 45 sec ------annealing Step 4: 72C for 1 min ------ elongation Repeat step 2 through step 4 for 30-40 cycles Step 6: 72C for 7 min ------ Final extension Step 7: 4C ∞ The amplified DNA will be store in a 4 °C fridge (up to one week) until gel electrophoresis. Electrophoresis Day 2 Preparation of Agarose Gel (work in groups for this step): You will run an agarose gel to check for successful amplification of the 16S rRNA gene. Only the samples that show one band of amplified DNA of the same length as the expected PCR product (~1500 bp), and whose control shows no band should be prepared for sequencing. Loading, running and analyzing the gel takes about an hour. Materials ​ Electrophoresis apparatus ​ Agarose ​ 500 mL (TAE) buffer ​ Loading dye (6X) ​ 1kb DNA ladder ​ P20 pipettes and tips ​ 10,000x GelRed ​ UV transilluminator Procedure a. Each group prepare the 1.0% Agarose gel as follow: In a 250mL flask mix 0.3 g agarose to ~30 mL TAE buffer and dissolve by heating in a microwave until just boiling (approx. 30 secs). Loosely plug opening with a kim-wipe before heating. b. Allow melted agarose to cool for a few minutes. c. After the agarose solution has cooled a little, add 3 ul 10,000x GelRed (1 mg/mL) in the hood and swirl to mix well. (see instructor) Then carefully pour into the casting tray and insert comb. Consider which end to place the comb. Allow the gel to solidify. Day 2 Running the Gel d. Carefully set the gel into electrophoresis apparatus, cover the gel completely with 1x TAE buffer. e. Each group will load 10 µL of 1kb molecular marker (ladder) into the first well. f. Work individually: Aliquot 1 ul of 6x loading dye and place the aliquot on a parafilm (you will need 3 drops, one for each sample). Combine 5 ul of each sample with the dye on the parafilm and mix using your pipette. Load the DNA-dye mixture into one of the available wells (ask the instructor for help). Make sure you change pipette tips between samples to prevent contamination. The left 20 µL of the PCR product will be remaining in each tube and should be stored on ice or at 4°C. Record in which lanes of the gel your samples are located. g. Once all samples have been loaded, close the lid on the apparatus, and attach the wires to the power supply (RED-RED, BLACK-BLACK). Be certain the DNA wells are oriented to run from (-) to (+) direction. h. Turn on the power supply, and run at 120V until the track dye has moved 2/3 the distance of the gel (about 45 min) i. Observe the gel on a UV transilluminator (use adequate protection against UV light!). The fluorescent bands show the presence of an amplified 16S rRNA gene if the PCR was successful. Take photographs with the gel documentation system. Photographs should be printed for each person in the group. Compare the bands of unknown organisms with the ladder in the same gel photo. Organisms of the same species should yield a similar banding pattern. How many bands do you see? What are the sizes (expressed in bp) of the PCR products of your unknowns? Is there a band in your negative control? Day 3 Clean Up PCR Products The amplified DNA has to be cleaned to remove excess primers and polymerase before sequencing. We will use the PureLink PCR Purification Kit which filters the DNA out with a solid-phase silica membrane and washes away undesired PCR reagents. Materials · Your PCR tubes · Sterile Eppendorf tubes · P1000 Pipettes and tips · PureLINK · Tabletop Microcentrifuge · Gloves · Ice bucket Procedure-Updated 9/26/23 7. Take your sample to the nanodrop to measure success. Make sure you enter your unknown number into the computer BEFORE you run your sample. It is best if you only run your sample once. The first person will blank the device with water. Each unknown will use 1 uL on the nanodrop. Please use a kimwipe to clean the nanodrop between samples. Once your sample is on the nanodrop, close the lid, then hit “measure”. You will see a number for your sample. Record this number on the sheet provided, along with your name and unknown number. Day 4 DNA sequence analysis Materials: · Your DNA sequences · Computer with internet connection Procedure: GenBank is the genetic sequence database of the National Institute of Health (NIH) and contains millions of sequence records. Scientists all around the world deposit DNA sequences at GenBank, including those derived from viruses, bacteria, and eukaryotic organisms. Therefore, this database is the ideal place to compare our new sequences with previously deposited sequences and identify our bacterial strains. 1. Trim the sequence to remove the "Ns" 2. Go to NCBI BLAST page (http://blast.ncbi.nlm.nih.gov/Blast.cgi). What does BLAST stand for? You will do a simple BLAST search using your DNA sequences, but you can do much more with BLAST. You are encouraged to work the Tutorials on the BLAST home page to learn more. 3. Select "nucleotide BLAST" (i.e. blastn) 4. Copy your sequence in the "Enter query sequence" field. 5. As you look down the BLAST page, you'll see an Options section. Under "Chose search net" (followed by a drop-down menu), select "nucleotide collection" 6. Scroll down and select "BLAST" 7. Be patient. Your results will appear in a few seconds/minutes. The program will find the most similar sequences that have been deposited in GenBank. 8. Results: As a rule of thumb, if your sequence has more than 97% similarity to a sequence deposited at the NCBI GenBank database, then it is a member of the same species. If the new sequence has more than 95% similarity to a previous sequence, then it is a member of the same genus. 9. Record your species on the board. If others have the same one, then add your initials after their record. 10. Use the internet to perform a search of the morphology, biochemistry, and physiology of the species you have identified. References: Don R, Cox P, Wainwright B, Baker K, Mattick J (1991). "'Touchdown' PCR to circumvent spurious priming during gene amplification". Nucleic Acids Res 19 (14): 4008. GenBank Overview (2010). Available online at: http://www.ncbi.nlm.nih.gov/genbank/ Johnson, J.L. (1984). In N.R. Kreig & J.G. Holt (Eds.), Bergey's Manual of Systematic Bacteriology, 1, 8-11. Baltimore: Williams. Lewin, B. (2010). Genes IX. Jones & Bartlett. http://biology.jbpub.com/book/genes/index.cfm Eldridge, M.L., Cadotte,M.W., Rozmus, A.E, and S.W. Wilhelm (2007). The response of bacterial functional groups to changes in available iron in the Eastern subtropical Pacific Ocean. The Journal of Experimental Marine Biology and Ecology 348:11-22.