Conventional PCR basics
Thermostable DNA polymerase is one key
All this amplification that sounds very sophisticated is actually done automatically by thermostable DNA polymerase, whose role is to replicate DNA. In order to prevent the random uncontrolled replication of all or unwanted parts of DNA in the sample, a set of primers are introduced into the reaction mixture: a forward primer that marks the beginning and a reverse primer that marks the end of a section of DNA that is to be amplified. By careful design of both primers (known sequence of nucleotides) we instruct the DNA polymerase exactly which part of the DNA it should amplify. And it does so. These short sections of DNA are usually a few hundred base pairs long in conventional PCR and only a few ten base pairs in qPCR.
Temperature cycling is the other key
In the beginning of PCR the DNA is heated up to 95°C so that the DNA is denaturated (single strands of DNA are obtained). In other words the DNA becomes exposed to DNA polymerase. But the DNA polymerase requires a double stranded DNA to start the polymerisation on the template DNA strand. Here is where the set of primers step in. The temperature is now lowered and the primers anneal to the complementary part of the DNA (according to A-T and G-C base pairing). Because there is an enormous excess of primers, they anneal to every specific target sequence on the template DNA no matter how many copies of the target there are. Now the DNA polymerase can start filling in the complementary DNA strand along the template DNA. The DNA polymerase replicates the short section of a DNA (also termed amplicon) until there is no more DNA template. It filled all the gaps it could. This was one cycle of PCR and there are twice as many specific parts of the DNA as were in the beginning.
Now the temperature is raised again so that the DNA is denaturated and, again, becomes exposed to DNA polymerase. This time, not only the DNA that was originally present in the sample but also the complementary strands which were synthesised in the first cycle represent the template for the DNA polymerase. During second cycle the number of specific sections of DNA is thus doubled (see image 1). This happens over and over again for 30 – 45 cycles. As you can see the only thing that is being changed during the cycles is temperature, everything else is done by itself (by DNA polymerase to be more specific). By increasing and decreasing the temperature we control the amplification of the DNA (see Image 2). Very simple, right? This cycling of the temperature is the reason why the PCR machines are also called thermal cyclers or PCR cyclers.
Image 1 shows one polymerase chain reaction (PCR) cycle. Cycling of temperature is the basis of PCR.
Image 2 shows a more detailed graphical represantation of PCR amplification (the first two cycles) as it is going on in the reaction tube.
Visualisation of results
In conventional PCR we can see the result of amplification only after the PCR is completed. Amplified products need to be visualised with another method such as agarose gel electrophoresis with intercalating fluorescent dyes (e.g. ethidium bromide; see Image 3).
Image 3 shows a picture (negative) of an agarose gel after electrophoresis stained with ethidium bromide under UV light. Columns "M" are molecular weight markers, each band representing a DNA fragment of a known length (shortest being on the bottom of the gel and longest being on the top of the gel). Samples "S1" to "S4" contain PCR products that were amplified during PCR . As you can see more than one PCR product was amplified in each sample (observe distinct bands of different lengths).
All this amplification that sounds very sophisticated is actually done automatically by thermostable DNA polymerase, whose role is to replicate DNA. In order to prevent the random uncontrolled replication of all or unwanted parts of DNA in the sample, a set of primers are introduced into the reaction mixture: a forward primer that marks the beginning and a reverse primer that marks the end of a section of DNA that is to be amplified. By careful design of both primers (known sequence of nucleotides) we instruct the DNA polymerase exactly which part of the DNA it should amplify. And it does so. These short sections of DNA are usually a few hundred base pairs long in conventional PCR and only a few ten base pairs in qPCR.
Temperature cycling is the other key
In the beginning of PCR the DNA is heated up to 95°C so that the DNA is denaturated (single strands of DNA are obtained). In other words the DNA becomes exposed to DNA polymerase. But the DNA polymerase requires a double stranded DNA to start the polymerisation on the template DNA strand. Here is where the set of primers step in. The temperature is now lowered and the primers anneal to the complementary part of the DNA (according to A-T and G-C base pairing). Because there is an enormous excess of primers, they anneal to every specific target sequence on the template DNA no matter how many copies of the target there are. Now the DNA polymerase can start filling in the complementary DNA strand along the template DNA. The DNA polymerase replicates the short section of a DNA (also termed amplicon) until there is no more DNA template. It filled all the gaps it could. This was one cycle of PCR and there are twice as many specific parts of the DNA as were in the beginning.
Now the temperature is raised again so that the DNA is denaturated and, again, becomes exposed to DNA polymerase. This time, not only the DNA that was originally present in the sample but also the complementary strands which were synthesised in the first cycle represent the template for the DNA polymerase. During second cycle the number of specific sections of DNA is thus doubled (see image 1). This happens over and over again for 30 – 45 cycles. As you can see the only thing that is being changed during the cycles is temperature, everything else is done by itself (by DNA polymerase to be more specific). By increasing and decreasing the temperature we control the amplification of the DNA (see Image 2). Very simple, right? This cycling of the temperature is the reason why the PCR machines are also called thermal cyclers or PCR cyclers.
Image 1 shows one polymerase chain reaction (PCR) cycle. Cycling of temperature is the basis of PCR.
Image 2 shows a more detailed graphical represantation of PCR amplification (the first two cycles) as it is going on in the reaction tube.
Visualisation of results
In conventional PCR we can see the result of amplification only after the PCR is completed. Amplified products need to be visualised with another method such as agarose gel electrophoresis with intercalating fluorescent dyes (e.g. ethidium bromide; see Image 3).
Image 3 shows a picture (negative) of an agarose gel after electrophoresis stained with ethidium bromide under UV light. Columns "M" are molecular weight markers, each band representing a DNA fragment of a known length (shortest being on the bottom of the gel and longest being on the top of the gel). Samples "S1" to "S4" contain PCR products that were amplified during PCR . As you can see more than one PCR product was amplified in each sample (observe distinct bands of different lengths).