Purpose: To be familiarized with the science and techniques used to identify different types of bacteria based on their DNA sequences. Background Information: The process begins with preparing a sample. Successful identification starts with using a sample that is considered to be good. The first step is to pick up a single colony and drop it into a microcentrifuge tube. A buffer is used to dissolve the cell wall in order to extract bacterial DNA. This step may take several hours. The proteolytic enzymes need to go before proceeding. Heating the sample in a water bath at 100 degrees Celsius denatures them.
Next, cellular debris is spun down in the centrifuge and appears as a pellet at the bottom. The DNA is contained in the liquid, which is then transferred to the tube. To continue the process, PCR amplification is conducted. One must add PCR Master Mix solution to the sample DNA to prepare the polymerase chain reaction. The mix contains water, a buffer to keep the correct pH for the reaction; large quantities of the four nucleotides; large quantities of oligonucleotide DNA primers; and a heat-stable DNA polymerase. At the same time, one will prepare negative and positive control reactions.
The positive contains positive control DNA while the negative contains sterile deionized water. Both contain the PCR solution. Once reaction tubes have been loaded onto the PCR machine, DNA replication starts. By doing this, one can know temperature, time remaining, cycle number, melt, anneal, and extend. The first step, melt, is to separate the two DNA chains in the double helix by heating the vial containing the PCR reaction mixture to 95 degrees Celsius for 30 seconds. The vial is cooled at 60 degrees Celsius. The final step, extend, is to allow the DNA polymerase to extend the copy DNA strand by raising the temperature to 70 degrees Celsius for 45 seconds.
Separation of the strands, annealing the primer to the template, and the synthesis of new strands all take less than two minutes. At the end of a cycle, each piece of DNA in the vial has been duplicated. The cycle can be repeated. The PCR product must be purified. First, insert the microconcentrator column of appropriate size into a collection tube. Then, add 400 uL of buffer and the entire PCR content to the column. Spin the column at 3,000 rpm in a fixed-angle centrifuge for 15 minutes.
The PCR product should be trapped in the column. Remove the collection tube and discard it. Next, invert the column and attach it to a new collection tube. Add 50 uL of buffer to the inverted column, which will loosen the DNA. Spin the inverted column at 3,000 rpm for 2 minutes to collect the sample, and then discard the column. One must then prepare for sequencing. The PCR tube now contains almost nothing but copies of the 16S rDNA. A set of 12 primers; six for each strand of the double-stranded DNA, is used. The PCR product from the previous step is added to the tube and another PCR is run. Each DNA strand binds the primer at one end and will have a fluorescence-tagged terminator at the other end.
DNA sequencing is now ready to complete. One has 12 tubes that contain the final PCR product, which is a mix of DNA pieces of variable length. All DNA pieces in each tube start with the same primer yet end with a different nucleotide tagged with a fluorescent marker. The individual DNA pieces are separated by using an automatic sequencer that performs gel electrophoresis on the DNA in each tube. The sequencer has a thin capillary tube attached at one end to a syringe mechanism that contains buffer solution. The tube is filled with buffer solution and the opposite end is inserted into one of the tubes containing.
Jourdyn Cowart Friday 1:00 the DNA pieces. An electric current is applied so that the end of the tube in contact with the DNA has a negative charge and the syringe end has a positive charge. DNA molecules move through the tube toward the positive charge end, with the smaller pieces moving faster than the ones that are larger. Optical detectors detect the color of the fluorescence. A complete set of DNA pieces with differentiated sizes were generated. The sequencer flushes out of the buffer from the tube, moves the tray, and runs the electrophoresis again.
This continues until all 12 tubes are examined. The resulting steps of the sequences are collated by a computer program to build the complete sequence of the gene. After the information for the sequence has been gathered, the computer builds the actual sequence by matching together the different pieces. One now has the 16S rDNA sequence for this bacterial species. To identify the sample, one must first view the data output from the sequencer. Then, one can go to the NCBI site to search. On the site’s BLAST page, one will enter the data and follow the instructions to obtain results.
Methods: Patient samples such as fluid from lymph node, stool, urine, blood, and sputum are used for the purpose of identifying possible pathogens. PCR contains everything that is necessary to carry out the polymerase chain reaction to amplify the 16S rRNA gene. It consists of water; a buffer to keep the mix at the correct pH for the PCR reaction; large quantities of adenine, cytosine, guanine, and thymine; large quantities of oligonucleotide DNA primers that bind the 16S rDNA region to initiate the replication process; and a heat-stable DNA polymerase that extends the copy DNA strand.
The desired DNA is separated from all others by an automatic sequencer that performs gel electrophoresis, which is a method to separate molecules based on differences in size. The sequencer has a thin capillary tube attached at one end to a syringe mechanism that contains a buffer solution. The tube is filled with buffer solution and the other end inserted into one of the tubes containing the DNA pieces. Then, an electric current is applied so that the end of the tube that is in contact with the DNA has a negative charge and the syringe has a positive charge. The DNA molecules move through the tube.
Near the syringe end, the capillary tube passes through a laser beam that excites the fluorescent markers, and optical detectors detect the color of fluorescence. The sequencer flushes out the buffer, moves the tray, and runs the electrophoresis again. When a person has the 16S rDNA sequence for a particular bacterial species, they can compare it with all other known 16S rDNA sequences for identification.
The BLAST nucleotide search engine is used by pasting the data in the box labeled “Enter accession number, gi, or FASTA sequence,” in the “Enter Query Sequence” near the top of the page. In the “Choose Search Set” section, Database: “Others (nr etc.)” and “Nucleotide collection (nr/nt)” should already be selected.
Then, click on the blue “BLAST” button and follow the instructions provided on the site to obtain results. Data: 1. Sample A is Bartonella henselae 2. Sample B is Escherichia coli 3. Sample C is Pseudomonas aeruginosa I entered the sequence into the NCBI search engine and clicked on the blue “BLAST” button, and it pulled up a color key for alignment scores that also included the name. Jourdyn Cowart Friday 1:00 Conclusion: I learned several processes from this virtual lab including PCR amplification, PCR purification, and DNA sequencing.