Part 1 - Sample Preparation
The key to a successful identification is to start with a "good" sample. Many pathogenic bacteria do not grow well on solid culture medium, making identification by traditional means difficult. (Why?) Even poorly growing bacterial cultures, however, can be identified with the methods used in this lab.

In this lab, you will act as a pathologist or perhaps a pathology lab technician at a well-equipped research hospital. Your task is to identify a bacterial sample received from a clinician.

Assuming that you have managed to grow bacterial colonies on a solid medium culture dish, the first step is to pick up a single colony and drop it into a microcentrifuge tube.

The process of extracting bacterial DNA consists of dissolving the cell wall with a digestive buffer (in the white-capped bottle) available as a commercial kit. The buffer contains proteolytic enzymes that "eat" the cell wall. This step may take several hours.

Since we will be using other enzymes in the next step, we need to get rid of the proteolytic enzymes before we can proceed. The enzymes are denatured by heating the sample in a water bath at 100°C. Next, the cellular debris is spun down in the centrifuge and appears as a solid deposit (pellet) at the bottom of the tube. The DNA is contained in the supernatant (the liquid), which is then transferred to the PCR tube.
Why /

Back to Part 1Note: Limitations of the traditional methods of identification

Over the years, a battery of tests has been developed to categorize and identify bacteria. Tests include staining and growing bacteria under a variety of conditions. Such procedures typically require vigorously and reliably growing bacterial cultures.

Many pathogens grow poorly on solid medium while others grow only in liquid culture, making identification through traditional techniques difficult or impossible. With the aid of molecular methods, however, these limitations can be overcome.

In addition, some species of bacteria cannot be differentiated from closely related species through traditional methods. For these species, molecular methods offer the only reliable and convenient means of identification. | |
What is PCR?

PCR (Polymerase Chain Reaction) is a technique that allows many copies of DNA to be made. It is one of the cornerstones of the current revolution in molecular genetics. Before the advent of PCR, making sufficient copies of DNA for sequencing was a tedious process. Now, PCR makes obtaining copies of DNA from a small original sample routine, with far-reaching consequences. For example, it made DNA finger-printing widely available, for use not only in forensic science for identifying potential suspects but also for use by behavioral biologists to trace paternities in a population of wild birds to monitor what type of behavior leads to success in mating.

In normal cells, the double-stranded DNA is unzipped with an enzyme to start the replication process. In PCR, single-stranded DNA is made by heating a chromosome fragment to 95°C. It is then cooled so that the primers anneal to the original DNA strands and DNA polymerase can bind and copy each strand. With repeated heating and cooling, millions and even billions of copies of DNA can be made in a few hours.

An interesting twist was the discovery of chemoautotrophic bacteria that live in the hydrothermal vents in the deep ocean floor. These creatures live in the vents where temperatures exceed 100°C; consequently, their DNA polymerases remain functional at such temperatures that would have denatured an ordinary creature's DNA polymerases. DNA polymerases isolated from these bacteria have made it possible to develop automatic thermocycler machines without the need to add new polymerases each cycle.

To obtain only the desired portion of the DNA, this lab uses oligonucleotide primers that specifically bind to regions flanking the 16S rRNA gene. (For further explanation, watch the PCR animation in the main lab.)

Step 1: Add Master Mix

To prepare the polymerase chain reaction (PCR), we will add the PCR Master Mix solution to our sample DNA. The PCR Master Mix solution (in the red-capped bottle) contains the following: water; a buffer to keep the mixture at the correct pH for the PCR reaction; large quantities of the four nucleotides adenine, cytosine, guanine, and thymine; large quantities of oligonucleotide DNA primers that bind the 16S rDNA region to initiate the replication process (What are primers?); and a heat-stable DNA polymerase that extends the copy DNA strand.

At the same time as the test reaction, we will prepare negative and positive control reactions. Instead of the sample DNA, the positive control reaction contains positive control DNA (the solution of 16S rDNA in the green-capped bottle) while the negative control reaction contains sterile deionized water. Both reactions contain the PCR Master Mix solution.

Step 2: Run PCR

Once the reaction tubes are loaded onto the thermocycler (the "PCR machine"), the automatic process of DNA replication starts (refer to the animation). The machine used in this lab has readouts that describe what is happening:

(from left to right: temperature, time remaining, cycle number, melt, anneal, and extend)

The temperature control is set up as follows: * Initial incubation step: 95°C for 10 minutes * 30 cycles of the following sequence of steps: * Melt: 95°C 30 seconds * Anneal: 60°C 30 seconds * Extend: 72°C 45 seconds * Final extension step: 72°C 10 minutes * Final step: 4°C store at this temperature
During each cycle, 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°C for 30 seconds. The primers cannot bind to the DNA strands at such a high temperature, so the vial is cooled to 60°C. At this temperature, the primers bind (anneal) to the single-stranded DNA. (The reason the two separated strands of DNA do not reanneal is that there is a large excess of primers in the solution; therefore, it's more likely for the DNA strands to bind to the primers instead of to each other.) The final step (Extend) is to allow the DNA polymerase to extend the copy DNA strand by raising the temperature to 70°C for 45 seconds.

The three steps—the separation of the strands, annealing the primer to the template, and the synthesis of new strands—take less than two minutes. Each is carried out in the same vial. At the end of a cycle, each piece of DNA in the vial has been duplicated. The cycle can be repeated 30 or more times, and each newly synthesized DNA piece acts as a new template. After 30 cycles, 1million copies of the initial DNA piece can be produced.
Part 3 - Purify PCR Product
The tube should now contain many copies of 16s rDNA, each about 1,500 base pairs (bp) long. At this time, it is prudent to run a gel to confirm that the PCR reaction worked. The gel should contain three lanes: one for the negative control (i.e., water), which should not have a product unless the water was contaminated; another for positive control (PCR product of a known DNA sequence) to make sure that the PCR itself worked; and the last lane for your sample.

If you are confident that the PCR worked, you can proceed to purifying the PCR product. Running a gel is actually one method of purification. Once the PCR product is in the gel, you can cut out the band corresponding to the PCR product and isolate the DNA from the gel. Nowadays, you can buy compact microfilters to filter the DNA from the PCR tube without running a gel. We will use such microconcentrator columns in our procedure: 1. Insert the microconcentrator column of appropriate size into a collection tube. 2. Add 400 µL of buffer to the column. 3. Add the entire PCR content (~100 µL) to the column. 4. Spin the column at 3,000 rpm in a fixed-angle centrifuge for 15 minutes. 5. The PCR product should be trapped in the column while the collection tube should contain all the primers, nucleotides, and other small compounds that we no longer need. Remove the collection tube and discard it. 6. Invert the column and attach it to a new collection tube. 7. Add 50 µL of buffer to the inverted column. This step should loosen the DNA from the column into the collection tube. 8. Spin the inverted column at 3,000 rpm for 2 minutes to collect the sample in the collection tube. Discard the column.
The final collection tube should now have many pieces of 1,500bp-long 16S rDNA, with a very small amount of longer DNA strands (which are contaminants).

Note: What is cycle sequencing?

In cycle sequencing, the first step is to use a thermocycler to create many copies of the target DNA but with one twist: you terminate the replication process at random places so the copies are all partial sequences with different lengths. The reaction mixture contains normal deoxynucleotides (A, C, G, and T) as well as some special dideoxynucleotides (A*, C*, G*, and T*) that are tagged with fluorescent markers. The fluorescent markers differ for each base, and are designed to fluoresce with different colors (G* is yellow, T* is green, for example). Dideoxynucleotides can substitute for normal deoxynucleotides during replication, but if such a substitution occurs by chance, that chain can no longer be extended, terminating that DNA strand. Dideoxynucleotides are thus called terminators. (See the pictorial representation in the lab animation.)

Let's use a specific example. To sequence a six-base-long single-stranded DNA (consisting of the sequence 3' A-C-G-T-T-G 5') with this method, you might start by generating pieces that are one to six bases long. Because DNA polymerase extends the copy DNA from the 5' end to the 3' end (i.e., from the 3' end to the 5' end of the template DNA), the pieces are going to be (remember that A pairs with T and C pairs with G)
T*
T-G*
T-G-C*
T-G-C-A*
T-G-C-A-A*
T-G-C-A-A-C*
In all of these pieces, the last (3' end) base is a dideoxynucleotide. Once you have these pieces, you can separate them based on their size by means of a very sensitive version of gel electrophoresis. Because each differently sized strand terminates in a specific base (for example, T-G-C* ends in a C*), it will fluoresce with a specific color. By looking at the fluorescent colors of DNA pieces of increasing size, you can compile the sequence for the complementary DNA (T-G-C-A-A-C), from which you can infer the original sequence.

Of course, for this to work, replication must start at the same place on the target DNA. In the above example, you do not want pieces such as G-C-A* (position 2-3-4) floating around. In addition, the normal sample contains double-stranded DNA, which means that you not only have the template we want (3' A-C-G-T-T-G 5') but also the complementary strand (5' T-G-C-A-A-C 3'). The complementary strand can create pieces such as G-T-T*, which is also undesirable. These problems are avoided by using primers that bind to specific known sequences of only one strand.In our example, instead of the six bases, consider a ten base strand with four more bases in the 3' end direction so the sequence is actually (3' T-G-T-A-A-C-G-T-T-G 5'). Using a primer sequence of 3' A-C-A-T, the replication will always start at the same place, and the resulting DNA pieces are going to be
A-C-A-T-T*
A-C-A-T-T-G*
A-C-A-T-T-G-C*
A-C-A-T-T-G-C-A*
A-C-A-T-T-G-C-A-A*
A-C-A-T-T-G-C-A-A-G*

Given that the incorporation of the terminator occurs by chance, it is difficult to make a very long DNA copy if you also want to make a short copy in the same reaction. Further, separating long chains by electrophoresis where the difference is only one base is difficult. Thus, to sequence a long section of DNA, many different primers are used, each in its own reaction tube. This way, many overlapping sections are sequenced in parallel and the result compiled to generate a complete sequence. | |
Prepare for Sequencing
The DNA sample has been purified; your PCR tube should now contain almost nothing but copies of the 16S rDNA. Now, we can prepare the sample for automatic sequencing. DNA sequencing technology is another area of molecular biology that has seen an impressive amount of refinement. The predominant method, illustrated here, is called PCR cycle sequencing. (Learn about cycle sequencing before proceeding.)

In this lab, we use a set of 12 primers; six for each strand of the double-stranded DNA. It is theoretically possible to use a single primer in PCR cycle sequencing, but it is not feasible for long sequences. With multiple primers, short, overlapping stretches of DNA are sequenced to obtain the complete sequence. In addition, it is not absolutely necessary to sequence both strands, although sequencing both strands generates redundant data, thereby reducing error. The exact number and location of primers used in a reaction depend on the availability of suitable primers. The primers used here are available from a commercial source and bind to conserved regions of the 16S rDNA gene. They should thus be able to bind to the sequence regardless of the bacterial source.

Each green and blue tube contains a "sequencing brew" consisting of buffers, primers (a different one in each tube), DNA polymerases, 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. This time the aim is not to produce identical copies of DNA but many copies of variable length. The animation illustrates what is happening in one tube containing the primer 651R. Each DNA strand binds the primer at one end and will have a fluorescence-tagged terminator at the other end.

Part 5 - DNA Sequencing
From the last step, you have 12 tubes that contain the final PCR product, a mix of DNA pieces of variable length. All DNA pieces in each tube start with the same primer but end with a different nucleotide tagged with a fluorescent marker (different color for each nucleotide A, T, G, C). What remains to be done is to separate the individual DNA pieces and identify the end nucleotide.

This is done by using an automatic sequencer that performs gel electrophoresis on the DNA in each tube. Gel electrophoresis is a method to separate molecules based on differences in size. The sequencer used in this lab has a thin capillary tube attached at one end to a syringe mechanism that contains a buffer solution. The tube is filled with the 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 in contact with the DNA has a negative charge and the syringe end a positive charge. Since DNA molecules are negatively charged, they start to move through the tube toward the positively charged syringe end, with the smaller pieces moving faster than the larger ones. Near the syringe end, the capillary tube passes through a laser beam that excites the fluorescent markers, and optical detectors detect the color of the fluorescence.

We can assume that a complete set of DNA pieces, all differing in size by exactly one nucleotide, were generated in the previous step. The smallest piece of DNA that has a fluorescent tag attached to it is the primer. This DNA fragment will travel faster than the other ones. By reading the color (in our example, red), we determine that the first nucleotide beyond the primer sequence is thymidine (T). The next smallest piece of DNA will fluoresce with the color (green) representing the next nucleotide in the sequence (A) and so on (see the animation). By reading the sequence of nucleotides based on their fluorescence as the DNA pieces pass through the laser beam, the sequence of the DNA can be reconstructed.

The sequencer automatically flushes out the buffer from the tube, moves the tray, and runs the electrophoresis again, repeating the program until all 12 tubes have been examined. The resulting sets of sequences are collated by a computer program to build the complete sequence of the 16S rRNA gene.
Back to Part 6

Note: The science behind sequence matching

Analysis of a newly isolated DNA molecule by comparing its sequence with all other known sequences in search of a match involves database searching and sequence alignment. The ultimate goal of this type of analysis is to determine whether the new sequence bears a significant degree of similarity (or homology) to another known sequence.

Perhaps the most important tool necessary for sequence matching is access to a comprehensive and up-to-date sequence database. GenBank, the EMBL nucleotide sequence database, and the DNA Database of Japan (DDBJ) are three partners in a longstanding collaboration to collect all publicly available sequence data. Sites in Bethesda, Maryland (USA), Hinxton (UK), and Mishima (Japan) exchange new sequence data and updates over the Internet everyday and make the information immediately available to everyone by e-mail, anonymous ftp, and the World Wide Web.

Next in importance is the computer program used to search the database. Several different mathematical programs allow two sequences to be compared with each other and determine the degree of similarity between them. The BLAST programs (BLAST is an acronym for Basic Local Alignment Search Tool) are among the most popular programs. They offer a good combination of speed, sensitivity, flexibility, and statistical rigorousness. (See interpreting BLAST search results.)

In what situations would a scientist search sequence databases? As an example, sequence matching can be used to determine whether a newly identified DNA sequence is part of a known gene. In the simplest scenario, if a new sequence is identical or almost identical (except for a few nucleotide changes) to that of a gene in the sequence database, it is reasonable to conclude that the new sequence is either part of the same gene or of a closely related gene. But what if two sequences which appear to be different share sections that are identical? How do you know whether the identical sections are due to chance or indicate some meaningful relationship between the two sequences? Sequence analysis using BLAST or another program provides a "similarity score" to help answer this question.

If the function of a particular DNA sequence is already known–for example, the 16S rRNA gene we have been working with in this lab–comparing its sequence with that of the same gene from another species of bacteria provides information about the evolutionary relationship between the two bacterial species. The assumption here is that the 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.

However, not all DNA sequences change at a constant rate over time. For example, it is not at all clear whether all organisms experience similar mutation rates from purely environmental factors (from increased UV exposure, for example). If the DNA sequence has or has had at some point in evolution a functional role, the rate of evolution and selection—which may be related to population size among other things—can affect its rate of change. And, in some cases, mutations are caused by deletions, insertions, and substitutions of long sequences of DNA rather than by single nucleotide changes. Finally, some sequences of DNA encode proteins with very specific structural requirements, and any change may prove unfavorable to the organism. Such sequences therefore do not tolerate change well and tend to remain the same for long periods of time. These are referred to as "conserved" regions. In contrast, sequences that can accommodate change more easily are referred to as "variable" regions.

Part 6 - DNA Sequence Analysis
Sample A - Fluid from Lymph Node
(See Samples section for more info)

After the sequence information has been gathered from all the reaction tubes, the computer builds the actual sequence by matching together different pieces. You now have the 16S rDNA sequence for this bacterial species, which can be compared with all other known 16S rDNA sequences for identification.

Learn about the science behind sequence matching.

There are many sequence databases in existence. Some databases are sold commercially as part of an identification kit. In this example, we will use the GenBank public database available through the National Library of Medicine. The matching algorithm is known as BLAST (Basic Local Alignment Search Tool). A direct match of the DNA sequence determines the exact bacterial species you have found. When the DNA sequence is not an exact match but a close match to another found in the sequence database, you need to assess whether it is a new species or a variation of an existing one.

Learn more about BLAST search results.

Now follow these steps to identify your sample:
Step 1: Click here to see the data output from the sequencer. Select the all data in the window and copy (ctrl-c on the PC or cmd-c on the Mac).

Step 2: Click here to go to the NCBI site to perform your search. (This will pop open a new window)

Step 3: On the NCBI site's BLAST page, paste your data (ctrl-v on the PC or cmd-v on the Mac) 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. When you are ready, click on the blue "BLAST" button. (updated October 21, 2007)

Step 4: Follow the instructions provided by the NCBI site to obtain your results.

Step 5: Come back to this page (which should remain open) and identify the bacteria in the lab window to the left.
Back to Part 2

Note: What are primers?

Primers are small pieces of DNA that bind to specific sequences—in this case, sequences within the 16S rRNA gene. In this lab, the two primers used, 27F and 1525R, bind to opposite ends of the gene, one on each DNA strand. Once the primers bind, DNA polymerase extends the DNA from the 5' end to the 3' end. The primers are selected so that as the two new copies are made, they overlap in the region of interest (see animation for illustration).

These primers are "universal," meaning that they bind to and thus copy the 16S rRNA gene from any bacterial species (except perhaps for the most unusual ones). This is because the sequences to which the primers bind are extremely similar among all bacterial species.

You might ask, If they are similar, how can you use them to identify different bacterial species? Doesn't that require uniquely different signatures? The answer is that some parts of a gene are extremely similar among different species (i.e., highly conserved) while others are highly variable. Universal primers bind to the highly conserved regions of genes so that they can be used to copy DNA from a variety of species of bacteria. The variable regions, which differ between species, are used for identification.