Chapter 6 PCR Identification of Microorganisms PDF

## Chapter 6 ### Identification of Microorganisms using Polymerase Chain Reaction (PCR) - **Introduction** In order to determine the relatedness of organisms from all domains of life (*bacteria, archaea*, and *eukaryotes*), it is important to find a trait that would be present in all living organ...

## Chapter 6 ### Identification of Microorganisms using Polymerase Chain Reaction (PCR) - **Introduction** In order to determine the relatedness of organisms from all domains of life (*bacteria, archaea*, and *eukaryotes*), it is important to find a trait that would be present in all living organisms. In the 1980s, Carl Woese suggested the use of DNA sequences of certain common genes. Such a molecular chronometer not only needed to be present in all organisms but also needed to have retained the same function. Woese proposed using a gene that encoded one of the RNA molecules found in ribosomes, the protein-RNA complexes on which proteins are synthesized in all prokaryotes and eukaryotes. Although there are differences in size between the ribosomes of prokaryotes (*70S*) and eukaryotes (*80S*), the sequences of the rRNA molecules (and their corresponding rDNA genes) from all sources contain regions that are very similar, allowing the alignment and comparison of these sequences. Further, the gene is small enough to be easily sequenced and large enough to contain enough information for genetic comparisons. Using extensive computer analyses of rRNA gene sequences, it has been possible to identify the so-called signature sequences, which are short oligonucleotides that are unique to certain groups of organisms. Not only are these signature sequences specific for each of the three domains of life, but they can also be specific within a domain or, a particular genus or even a single species. The specificity of the signature sequences is useful for many purposes. For example, signatures can be used for quickly placing a newly isolated or misclassified microorganism into its correct phylogenetic group. Using the sequence of 16S rRNA genes isolated from DNA of environmental samples allows us to define the composition of a microbial community without relying on the ability to culture all organisms present in these environments. ### Ribosomes of prokaryotes (70S) and eukaryotes (80S) This is a diagram of a prokaryotic and eukaryotic ribosome. - **Prokaryotic Ribosome:** The ribosome is composed of a 30S subunit and a 50S subunit. The 30S subunit contains a 16S rRNA molecule. The 50S subunit contains a 23S rRNA molecule and a 5S rRNA molecule - **Eukaryotic Ribosome:** The ribosome is composed of a 40S subunit and a 60S subunit. The 40S subunit contains an 18S rRNA molecule. The 60S subunit contains a 28S rRNA molecule, a 5.8S rRNA molecule, and a 5S rRNA molecule. ### Polymerase Chain Reaction (PCR) PCR was developed by the Nobel Prize winner Kary Mullis and associates in 1985 and is essentially a method that uses the smallest amounts of samples and by which one can replicate a specific segment of DNA (a gene or a smaller segment) millions of times while the rest of the DNA is not replicated. The reagents needed for amplification are: - the target DNA sample - two oligonucleotide primers that can complement the base pairing at the opposite ends of the target DNA - a suitable DNA polymerase (*Taq DNA polymerase*) - the four deoxyribonucleoside triphosphates dATP, dTTP, dGTP and dCTP (*dNTPs*) - Mg++ ions - **Heat-stable DNA polymerase** - *Taq DNA polymerase* was isolated from the *Thermus aquaticus*. - *Taq DNA polymerase* is stable at the high temperatures (~95°C) used for denaturing DNA. To perform the PCR, the reagents are mixed in a thin-walled tube and placed in an instrument called a *thermocycler*. What this machine does is that it changes the temperature very quickly according to a set of instructions. The changes in the temperature cause: 1. **Denaturation** of the target DNA strands 2. **Annealing** of primers to the separated strands 3. **Primer extension**. These 3 actions constitute a cycle. Many such cycles can be performed quickly to obtain a large number of copies of the target DNA. ### PCR Components and Process This is a diagram showing the components of the PCR reaction and the steps involved in one PCR cycle. There are four key components: - **DNA Sample:** The DNA sequence to be amplified. - **Primers:** Short single-stranded DNA sequences that are complementary to the target DNA sequence. They bind to the target DNA sequence and provide a starting point for DNA polymerase to begin synthesizing new DNA strands. - **Nucleotides:** Building blocks of DNA (i.e. adenine, guanine, cytosine, and thymine) - **Taq Polymerase:** A heat-stable DNA polymerase that can withstand the high temperatures required for PCR and can synthesize new DNA strands from the primer binding sites on the target DNA sequence. **PCR Process (ONE Cycle):** 1. **Denaturation** (95°C): The DNA strands are separated by heating the reaction mixture to a high temperature, breaking the hydrogen bonds between the complementary base pairs 2. **Annealing** (55°C): The reaction is cooled to allow the primers to anneal or bind to the complementary sequences on the separated DNA strands. 3. **Extension** (72°C): The DNA polymerase extends or elongates the new DNA strand from the primer binding site by adding nucleotides to the complementary sequence on the template strand. ### Agarose Gel Electrophoresis of the PCR Product Suppose we take a long piece of a DNA molecule and digest it with a restriction enzyme. It is important for us to know how many fragments are obtained and what their sizes are. Fragments can be separated on the basis of their size, shape, and charge by the technique of gel electrophoresis. The cut DNA is placed at one end of an agarose gel and subjected to an electrical current. Small fragments will move fast in the agarose, while large fragments move at a slower rate. In this way, fragments are separated into distinct bands, the approximate sizes of which can be estimated if a sample of fragments of known sizes is run alongside the unknown sample. In general, electrophoresis is a method used to separate cell fractions, proteins, nucleic acids, or even intact chromosomes. There are many different protocols used in electrophoresis but all take advantage of an inert support matrix (*e.g.* starch, agarose, polyacrylamide) and an electrical field. Molecules move in this electric field. The solid support that we will be using here is agarose, supplied as a polysaccharide powder. The agarose bed serves as a molecular sieve to separate the molecules according to their size, shape, and net ionic charge. DNA is negatively charged at neutral pH and thus moves towards the anode. When mixed with water, boiled, and then cooled, agarose will turn into a gel-like substance. This gel is a complex network of fibrils (of polysaccharide bonded together by hydrogen bonds) through which the DNA fragments must pass. The distance between the fibrils or the pore size is determined by the concentration of agarose used; *i.e.*, a 20% agarose would hardly let even very small DNA fragments to pass through while a 0.2% agarose is rather flimsy and can let very large DNA fragments travel through rather easily. So, controlling the concentration of agarose based on the sizes of the DNA molecules is important. Electrophoresis is conducted in a Plexiglas apparatus composed of a middle section where the agarose gel is placed and two buffer compartments or tanks at either end, connected by platinum wires to the current. ### Gel Electrophoresis This is a diagram illustrating the gel electrophoresis process, which is used to separate DNA fragments by size. 1. **Place DNA restriction fragments** in the well of an agarose or polyacrylamide gel. 2. **Apply electric field:** The negatively charged DNA fragments will migrate through the pores of the gel towards the positive electrode. The smaller fragments will move through the pores faster than the larger fragments. 3. **Subject to autoradiography or incubate with fluorescent dye:** The separated DNA fragments can be visualized using autoradiography or by incubating with fluorescent dyes that intercalate between the bases of the DNA molecules. ### Separating Restriction Fragments or PCR products, I and II This is a diagram illustrating the setup for separating restriction fragments or PCR products using agarose gel electrophoresis. - The process is initiated by loading the DNA samples in the wells of the agarose gel. - The gel is then placed in an electrophoresis chamber filled with buffer solution. - An electric current is applied, causing the negatively charged DNA fragments to migrate through the gel matrix. - The rate of migration depends on the size of the fragments and the concentration of the agarose gel, causing the fragments to separate according to their sizes. - **Agarose Electrophoresis Loading** - The electrical current carries negatively-charged DNA through the gel towards the positive (red) electrode. - **Agarose Electrophoresis Running** - The agarose gel sieves the DNA fragments according to size, with smaller fragments moving farther than large fragments. We add a marker dye (*bromophenol blue*) to the samples to be able to visually follow the front. After the front has traveled the length of the field, the gel is taken out and stained with an intercalating dye such as ethidium bromide (*EtBr*), SYBR Gold or Red Safe. *EtBr* and SYBR Gold are dyes that fluoresce under ultraviolet radiation and have a very high affinity for the double-stranded DNA molecule. For example, if we place the gel in a solution of *EtBr* for a short time, take it out, wash it with water, and then irradiate it with ultraviolet, the DNA molecules in the gel fluoresce, and we can photograph the gel to obtain a permanent record. The positions of the DNA fragments appear as clear bands in the gel and the photograph. By comparing each band with a standard, run in parallel in the gel, we can approximate the size of each fragment. Unfortunately, *EtBr* is a highly mutagenic and carcinogenic agent and extreme care should be practiced in its handling. Recently new dyes (*i.e.* Red Safe) have been produced by chemical companies that are quite good in resolving electrophoretic bands while they are not as toxic as *EtBr*. ### Agarose Gel Stained with Ethidium Bromide (EtBR) to Visualize the DNA (PCR product or Restriction DNA fragments) This is a diagram of an agarose gel stained with ethidium bromide that has been used to separate DNA fragments by size. - The gel shows different bands of DNA fragments separated according to size (1000 bp, 700 bp, 600 bp, 500 bp). - The **correct PCR product** is identified as a band on the gel. - A **DNA standard ladder marker** is included on the gel to provide a reference for the size of the DNA fragments. - **When the blue band which loaded with the bromophenol blue (marker dye) has reached halfway to the end of the gel, turn off the power supply, wear rubber gloves, and place the gel over a UV illuminator and let your TA take a photograph.** - **The 100 base pair (bp) standard ladder is used to determine the size (length) of a DNA band on a gel.** ### Identification using 16S rRNA gene sequence analysis Depending on the goal of the analysis, different primers may be needed for the amplification of the 16S rRNA gene. To determine the presence of a specific organism in an environmental sample, one can choose primers that will only bind to signature sequences of this organism. However, if the question is rather how diverse the bacterial community is (*for example*, from a swab sample of your mouth), one should use universal primers that will bind to conserved sequences of the 16S ribosomal-RNA gene of the domain *Bacteria*. Depending on the primer sequences and the length of the expected product, the conditions for the PCR will vary. ### Using BLAST to Identify the Species Based on its rRNA Gene Nucleotide Sequence BLAST stands for Basic Local Alignment Search Tool. It is essentially a search engine that searches a database of DNA sequences at very high speed. [http://ncbi.nlm.nih.gov/Education/blasttutorial.html](http://ncbi.nlm.nih.gov/Education/blasttutorial.html) - For a thorough tutorial on how to use BLAST. [http://www.ncbi.nlm.nih.gov/BLAST/blast_overview.html](http://www.ncbi.nlm.nih.gov/BLAST/blast_overview.html) - An overview of BLAST is found at: It should be mentioned that BLAST is as effective as the data it contains. For example, if the sequence of a specific species is not contained in the database, BLAST cannot produce an accurate result. In such a case, many close relatives would be shown at the end of the search. However, most of the time, BLAST results in correct identification of the genus and the species. There are occasions where some species are extremely close genetically (*at least for the 16 S rRNA gene*) to give a 100% definite outcome. There are also times when the genus is given, and the species name is not completely certain. So, combining the search results with other diagnostic tests (*such as the colony morphology*) may become very important in deducing the correct answer. What follows is just a short summary of the steps that are required for identifying your unknown. - Download your unknown 16S rRNA gene sequence from the BlackBoard Website to your computer. If you did not get a good sequence, there would be some extra files on the BlackBoard that you may download instead of yours. Your TA will give you more instructions in this regard. - Open your browser (*Netscape*, *Internet Explorer*, *etc.*) to: [http://www.ncbi.nlm.nih.gov/BLAST](http://www.ncbi.nlm.nih.gov/BLAST) - Under "Basic BLAST" click on "nucleotide blast". A new page will open up. - This page has several components. These components and the parameters that we choose are as follows: - **Enter Query Sequence:** Either paste your sequence directly in the box given or press the "Browse" button and find your file on your computer to be used in the search. You can also give a name to your search in the "Job Title" box. - **Choose Search Set:** Since our search does not involve human or mouse databases, click on the "Others (nr etc.)" button. Click on the drop-down menu and choose "16S ribosomal RNA sequences (Bacteria and Archaea)”. - **Next in the" Exclude" option, place a checkmark in the box for "Uncultured/environmental sample sequences". Leave the optional "Organism" and "Enterez Query" as they are.** - **Program Selection: Click on the radio button next to "Highly similar sequences (megablast)".** - **Finally press the big blue BLAST button on the left panel to get to the results. However, if you find that you need to find more similar species, first press "Algorithm parameters" located underneath the BLAST button and increase the number of results from the default 100 to 250 or even 500, then press the BLAST button.** - After pressing the BLAST, a new page appears that may get refreshed a few times. Finally, a final page will appear that will give you an ID # (*Query ID*), the number of nucleotides present in your sequence (*Query Length*), and some other information. Underneath you will see red bars, each of which represents a matching species. Scrolling down further, you can see the species names that match your unknown. The best match is at the top, and the matching becomes less accurate as you go down. Pressing on the "Accession" brings up a rather comprehensive description of the matching species, while pressing the "Max Score", shows the actual matching of nucleotides between your sample and the matching species. The species with the highest Max Score is usually the species that should be taken as your unknown result. However, sometimes a few species have the same Max Score, and then you may need to read about the differences among them by consulting the Bergey's Manual and checking the described characteristics against your unknown. Make a printout of the first 2-4 pages of these results and include it in your report. ### Rapid microorganism identification by DNA amplification and sequencing This diagram illustrates the steps involved in rapid microorganism identification by DNA amplification and sequencing: 1. **Extract DNA:** DNA is extracted from the unidentified microorganism. 2. **Amplify a marker (16S rRNA gene):** A marker (16S rRNA gene) that serves as an organism barcode is amplified. 3. **Read the amplified DNA barcode by DNA sequencing:** The amplified DNA barcode is read by DNA sequencing. 4. **Align the DNA barcode with all others in the database:** The DNA barcode is aligned with all others in the database. 5. **Identify the most similar microorganism and infer the relationship to other microorganisms:** Comparison of the aligned DNA barcodes is used to identify the most similar microorganism and to infer the relationship to other microorganisms. ### BLAST Results This is a screenshot showing the results of a BLAST search using the 16S rRNA gene, which indicates highly similar species to *Erwinia amylovora* and its accession numbers. This highlights the use of BLAST in identifying organisms based on their genetic sequences.