Information

If we add only 1 primer in PCR


What would happen if we add only one primer, say forward primer, to PCR?

(Image Credits: Wikipedia) As it is clear from the image that we need both forward and reverse primers to get it working (Unless we have a sequence such that a single primer can work as both forward and reverse). I have read online here that including a single primer will result in linear amplification but I think there should be no amplification at all. Can you help me resolve my doubt?


I think the following should happen: There would be no amplification but we will get n copies of single stranded DNA after n cycles and the only double stranded DNA with started with but with a new complementary strand.


Using 1 primer will results in amplifying the original sequence in each cycle in a linear fashion: the primer will guide a single run of the polymerase. In opposed to the "chain reaction" in PCR which is exponential. Therefore, with one primer you linear amplification of say x40 times the original number of molecules you started with, whie with two primers you get exponential (~240) amplification. You right that with one primer, the amplification is neglectable, almost invisible.


Polymerase Chain Reaction

David P. Clark , Nanette J. Pazdernik , in Molecular Biology (Second Edition) , 2013

Modified PCR Techniques Solve Problems

Degenerate primers are used when only part of the target sequence is known. In these primers, a mixture of bases is present in a given position on the primers. In this way, there is a chance of having at least one sequence in the mix of degenerate primers that is able to bind the target.

DNA primers for PCR do not have to be completely complementary to their target sequences. If most of the bases in the primer can bind the target, then that is usually sufficient for PCR. Because of this, primers are often engineered containing restriction enzyme sites or other sequences on their 5´ end. The end result of this is that the products of PCR contain restriction enzyme sites (for example), which can be used in subsequent techniques.

Longer target sequences can be amplified by varying the time of elongation and the type of DNA polymerase.

Long-range PCR is a variation on the normal amplification method of PCR that is used when target sequences are extremely long. Several simple modifications are made to PCR so that these targets can be amplified.

Taq polymerase does not proofread. In longer targets, if Taq polymerase is the only enzyme used, the possibility of accumulating mutations is greater. However, Pfu and other polymerases do proofread. So using a mixture of Taq and Pfu increases the fidelity of the final PCR product.

Additional changes to the reaction times of the process are also paramount in production of a long target. The elongation time is often extended from 1–2 minutes to 10–20 minutes each cycle. This allows the polymerase to continue along the entire length of the long target. Additionally, the denaturation step is decreased to just a few seconds to help protect some of the nitrogenous bases from constant exposure to heat, which could damage the purines (guanine and adenine).

Inverse PCR amplifies unknown sequences by creating a circular template DNA.

During inverse PCR, a restriction enzyme cuts on both sides of the known sequence. The two sticky ends are then ligated back together to generate a circular molecule. The primers for this variation of PCR are designed to extend outward from each other as opposed to elongated towards each other. Consequently, during PCR the polymerase extends one counterclockwise and the other clockwise. In this way, the unknown regions can be amplified.


PCR primers are designed to only replicate the N gene sequence of the viral genome. Part of the N sequence we want to amplify is shown below. Typically, you design two primers, one to bind to each strand of the dsDNA. Copies are made from each strand so you get twice as much DNA from the PCR process. Potential primer locations are noted by the nucleotide sequences shown below. The PCR needs to make copies of the nucleotides shown in the middle (“87 nucleotides”). Remember the direction that DNA polymerase synthesizes new DNA strands. Select the two locations for the primers to bind and then fill in the correct sequence below the DNA sequence shown. You should have selected one location on each strand. Indicate the direction that the DNA polymerase (Taq polymerase) will move after binding to the primers in the attached image

PCR primers are designed to only replicate the N gene sequence of the viral genome. Part of the N sequence we want to amplify is shown below. Typically, you design two primers, one to bind to each strand of the dsDNA. Copies are made from each strand so you get twice as much DNA from the PCR process.

Potential primer locations are noted by the nucleotide sequences shown below.

The PCR needs to make copies of the nucleotides shown in the middle (“87 nucleotides”).

Remember the direction that DNA polymerase synthesizes new DNA strands. Select the two locations for the primers to bind and then fill in the correct sequence below the DNA sequence shown. You should have selected one location on each strand.

Indicate the direction that the DNA polymerase (Taq polymerase) will move after binding to the primers in the attached image

fullscreen

DESIGN AND IMPLEMENTATION

To maximize the utilization of the server resources, PCRTiler has been implemented as a multi-threaded application that designs as many primer pairs concurrently as the server has processors. Independent tiling requests are queued until the currently executing tiling job is finished. Users providing an email address will be notified when their request has finished processing. Others will have to use the link provided on the submission confirmation page to view their result.

To promote fair use of the system, the total number of primer pairs that can be designed in a single request is limited to 200, and the maximum duration of a tiling job is set to three hours. Users exceeding those limits can still use PCRTiler, either by installing the standalone PCRTiler application on their personal computer, installing the server version and disabling the limit, or splitting their large request into smaller regions.

PCRTiler will gracefully recover from server restarts. As soon as new tiling requests are submitted to the server, they are compressed and then saved to disk. In the event that the server is restarted, PCRTiler will transparently recover the queued tiling requests, preserving their original order, and resume execution of the run that was aborted.

Software and hardware requirements

PCRTiler requires the Java Runtime Environment (JRE) v1.6.0 and Tomcat 6 running on a computer using the Linux operating system. It should theoretically also run on any combination of platforms and operating systems for which implementations exist for the JRE, Tomcat 6, Primer3 and BLAST binaries, but this has not been tested and therefore is unsupported. During testing, we have validated that it behaves properly when viewed with the latest versions of Firefox, Safari and Internet Explorer.

The performance of PCRTiler is primarily dependent on the available memory. In our experience, for acceptable performance, you need enough memory for the BLAST database (800 MB for Homo sapiens, 5 MB for most bacteria), plus a maximum of 1 GB for PCRTiler. Therefore, 2 GB of memory should be enough. This amount of memory is commonly included in recent workstation computers and laptops. PCRTiler is a multi-threaded application, so it will make use of all available CPU cores, accelerating primer design proportionately to the number of cores. The PCRTiler server currently runs Mandriva Linux 2010 on a dedicated Quad-core Intel machine clocked at 2.4 Ghz with 4 GB of RAM. Including the BLAST databases of all 1169 genomes, PCRTiler requires <15 GB of hard disk space.

Standalone version

In addition to the server version, we provide a standalone Java-based application, which includes a graphical user interface and the same one-click genome management feature as the server version. It also handles all aspects of downloading genomes from GenBank and the creation of BLAST databases. Since the standalone and server versions share much of the same code base, they both provide the same functionality. However, the standalone version uses the resources of the client computer. Using the standalone version is the easiest option for most users who want to run PCRTiler locally. Please note that the standalone version does not require Tomcat. To date, it has been shown to work correctly on Linux i386, Windows Vista and Windows XP.

Data retention

PCRTiler results are kept on the server for 14 days. However, users have the option of deleting their result file from the server immediately using the appropriate button on the result page. Users that would like to hold on to a PCRTiler result for a longer time period can download the raw result file from the website, which can be viewed using the standalone version of PCRTiler.


Why do we use a negative control in PCR?

PCR works off of a template DNA. Lets say you are testing for HIV (HIV is an RNA virus, but when it goes into a cell, it gets turned into DNA. so there will be HIV DNA in an infected cell). The primers you use will make a product (amplicon) that corresponds to part of the HIV DNA. If you see this amplicon, then you have the HIV sequence present. but if you don't have a negative control, you may have contamination.

PCR is extremely senstitive. There are many solutions used in PCR (water, buffer, dNTPs, enzyme). and all of them can easily get contaminated with DNA from other samples, or even from the amplicon that was made in the reaction you did yesterday. So if you have Patient X's sample of DNA and you are checking for HIV by PCR, the PCR primers may make a product off of the HIV DNA in Patient X's DNA sample (if that person has HIV), or it may make it off of contamination. But you can't tell if it is from contamination or from HIV in the patients DNA.

So you run a water control. Tube 1 you place all the components of the reaction, and for the DNA you only add water. This is the negative control. NOTHING should amplify here. In Tube 2 you put all the reaction components and Patient X's DNA. If you get a product here, (and nothing in Tube 1), Patient X probably has the HIV DNA in his/her DNA. If you get a product in both Tube 1 and Tube 2, you have a contamination problem and you can't tell if the HIV in the Patient's sample is from the disease or from contamination.


The Polymerase Chain Reaction (PCR) is a method of DNA replication that is performed in a test tube (i.e. in vitro). Here &ldquopolymerase&rdquo refers to a DNA polymerase enzyme extracted and purified from bacteria, and &ldquochain reaction&rdquo refers to the ability of this technique produce millions of copies of a DNA molecule, by using each newly replicated double helix as a template to synthesize two new DNA double helices. PCR is therefore a very efficient method of amplifying DNA.

Besides its ability to make large amounts of DNA, there is a second characteristic of PCR that makes it extremely useful. Recall that most DNA polymerases can only add nucleotides to the end of an existing strand of DNA, and therefore require a primer to initiate the process of replication. For PCR, chemically synthesized primers of about 20 nucleotides are used. In an ideal PCR, primers only hybridize to their exact complementary sequence on the template strand (Figure (PageIndex<3>)).

Figure (PageIndex<3>): The primer-template duplex at the top part of the figure is perfectly matched, and will be stable at a higher temperature than the duplex in the bottom part of the figure, which contains many mismatches and therefore fewer hydrogen bonds. If the annealing temperature is sufficiently high, only the perfectly matched primer will be able to initiate extension (grey arrow) from this site on the template. (Original-Deyholos-CC:AN)

The experimenter can therefore control exactly what region of a DNA template is amplified by controlling the sequence of the primers used in the reaction.

To conduct a PCR amplification, an experimenter combines in a small, thin-walled tube (Figure (PageIndex<4>)), all of the necessary components for DNA replication, including DNA polymerase and solutions containing nucleotides (dATP, dCTP, dGTP, dTTP), a DNA template, DNA primers, a pH buffer, and ions (e.g. Mg 2+ ) required by the polymerase. Successful PCR reactions have been conducted using only a single DNA molecule as a template, but in practice, most PCR reactions contain many thousands of template molecules. The template DNA (e.g. total genomic DNA) has usually already been purified from cells or tissues using the techniques described above. However, in some situations it is possible to put whole cells directly in a PCR reaction for use as a template.

Figure (PageIndex<4>): A strip of PCR tubes (Wikipedia-madprime-GFDL)

An essential aspect of PCR is thermal-cycling, meaning the exposure of the reaction to a series of precisely defined temperatures (Figure (PageIndex<5>)). The reaction mixture is first heated to 95°C. This causes the hydrogen bonds between the strands of the template DNA molecules to melt, or denature. This produces two single-stranded DNA molecules from each double helix (Figure (PageIndex<6>)). In the next step (annealing), the mixture is cooled to 45-65°C. The exact temperature depends on the primer sequence used and the objectives of the experiment. This allows the formation of double stranded helices between complementary DNA molecules, including the annealing of primers to the template. In the final step (extension) the mixture is heated to 72°C. This is the temperature at which the particular DNA polymerase used in PCR is most active. During extension, the new DNA strand is synthesized, starting from the 3' end of the primer, along the length of the template strand. The entire PCR process is very quick, with each temperature phase usually lasting 30 seconds or less. Each cycle of three temperatures (denaturation, annealing, extension) is usually repeated about 30 times, amplifying the target region approximately 2 30 -fold. Notice from the figure that most of the newly synthesized strands in PCR begin and end with sequences either identical to or complementary to the primer sequences although a few strands are longer than this, they are in such a small minority that they can almost always be ignored.

Figure (PageIndex<5>): Example of a thermalcycle, in which the annealing temperature is 55°C. (Original-Deyholos-CC:AN)

Figure (PageIndex<6>): PCR with the three phases of the thermalcycle numbered. The template strand (blue) is replicated from primers (red), with newly synthesized strands in green. The green strands flanked by two primer binding sites will increase in abundance exponentially through successive PCR cycles. (Wikipedia-madprime-GFDL)

The earliest PCR reactions used a polymerase from E. coli. Because the high temperature of the denaturation step destroyed the enzyme, new polymerase had to be added after each cycle. To overcome this, researchers identified thermostable DNA polymerases such as Taq DNA pol, from Thermus acquaticus, a thermophilic bacterium that lives in hot springs. Taq, and similar thermostable polymerases from other hot environments, are able to remain functional in the repeated cycles of amplification. Taq polymerase cannot usually amplify fragments longer than about 3kbp, but under some specialized conditions, PCR can amplify fragments up to approximately 10kbp. Other polymerases, either by themselves or in combination with Taq, are used to increase the length of amplified fragments or to increase the fidelity of the replication.

After completion of the thermalcycling (amplification), an aliquot from the PCR reaction is usually loaded onto an electrophoretic gel (described below) to determine whether a DNA fragment of the expected length was successfully amplified or not. Usually, the original template DNA will be so dilute that it will not be visible on the gel, only the amplified PCR product. The presence of a sharp band of the expected length indicates that PCR was able to amplify its target. If the purpose of the PCR was to test for the presence of a particular template sequence, this is the end of the experiment. Otherwise, the remaining PCR product can be used as starting material for a variety of other techniques such as sequencing or cloning.

An Application of PCR: the StarLink Affair

PCR is very sensitive (meaning it can amplify very small starting amounts of DNA), and specific (meaning it can amplify only the target sequence from a mixture of many DNA sequences). This made PCR the perfect tool to test whether genetically modified corn was present in consumer products on supermarket shelves. Although currently (2013) 85% of corn in the United States is genetically modified, and contains genes that government regulators have approved for human consumption, back in 2000, environmental groups showed that a strain of genetically modified corn, which had only been approved for use as animal feed, had been mixed in with corn used in producing human food, like taco shells.

To do this, the groups purchased taco shells from stores in the Washington DC area, extracted DNA from the taco shells and used it as a template in a PCR reaction with primers specific to the unauthorized gene (Cry9C). Their suspicions were confirmed when they ran this PCR product on an agarose gel and saw a band of expected size. The PCR test was able to detect one transgenic kernel in a whole bushel of corn (1 per 100,000). The company (Aventis) that sold the transgenic seed to farmers had to pay for the destruction of large amounts of corn, and was the target of a class action law-suit by angry consumers who claimed they had been made sick by the taco shells. While no legitimate cases of harm were ever proven, and the plaintiffs were awarded $9 million, of which $3 million went to the legal fees, and the remainder of the judgment went to the consumers in the form of coupons for taco shells. The affair damaged the company, and exposed a weakness in the way the genetically modified crops were handled in the United States at the time.