The molecules created in a PCR reaction are not all the same length. PCR necessitates a thorough understanding of the DNA sequence to be examined. You just finished 11 terms of study! Take a break and have some fun by taking a test or two.
Polymerase chain reaction (PCR) is a commonly used method for rapidly producing millions to billions of copies (full or partial copies) of a given DNA sample, allowing scientists to take a very little sample of DNA and amplify it (or a portion of it) to a big enough number to investigate in depth. The PCR process consists of two main steps: denaturation and annealing/ligation.
In the first step, known as denaturing, the double helix structure of DNA is disrupted so that the two strands will be able to separate from one another. This step is usually done by placing the sample in a heat-liable buffer that contains chemicals which help break down hydrogen bonds between complementary bases (the building blocks of DNA) thus freeing up space for other chemicals to come into contact with these bases. As well as breaking down hydrogen bonds, these chemicals can also help stabilize the single strand of DNA. Once the DNA has been denatured, it is cooled down quickly to prevent further melting of the base pairs.
In the second step, known as annealing/ligation, the sample is returned to room temperature or slightly below it. At this point, primers will anneal to their respective locations on the denatured DNA and make them ready to be copied. Next, enzymes called polymerases copy the DNA fragment using the primers as starting points, adding nucleotides to form long chains.
The polymerase chain reaction (PCR) is a method for "amplifying" tiny portions of DNA. The term "amplifying" means that multiple copies of the piece of DNA are made so that enough material is available for testing or for use in gene therapy projects.
What does PCR stand for? Polymerase Chain Reaction.
How did they come up with PCR? Dr. Kary Mullis invented PCR while working at Cetus Corporation in California. He used the procedure to discover how many human genes there are, so that scientists could better understand disease processes and develop drugs that target specific genes. The technique has since been adopted by researchers around the world for a wide variety of applications.
What types of materials can I use for PCR? You can use any organic material as long as it contains nucleic acids. This includes proteins, cellulose, and all plant and animal tissue. Synthetic materials such as glass, plastic, metal, and quartz have also been used successfully with proper cleaning.
What forms of energy are needed for PCR? PCR requires heat to break down DNA into fragments suitable for recycling.
PCR was initially designed to amplify small parts of a larger DNA molecule (Saiki et al., 1985). Target DNA, a thermostable DNA polymerase, two oligonucleotide primers, deoxynucleotide triphosphates (dNTPs), reaction buffer, and magnesium are commonly used in amplification reactions. The primer sequences must contain complementary regions that match the target sequence exactly or nearly so. Complementary base pairing is necessary for efficient annealing of the primer to its matching site on the template strand.
For each round of replication, the enzyme makes a copy of itself using one of the primers as a guide. Thus, the original template DNA is copied many times over, and eventually, there will be enough replicated material that it can be seen by agarose gel electrophoresis. If you were to repeat this process many times, you would end up with a mixture of different lengths of DNA fragments originating from the original template.
The short fragment present in the mixture is called product DNA. It contains copies of both primers, which allow it to be used as a template for further replication steps. As more than one copy of the primer can bind to the template, the product becomes saturated with primers. This allows additional primers to join the reaction at any point, creating long chains that can be separated from the reaction mixture and used as a template for further amplification.
Real-Time PCR is designed to capture data while the reaction is running, which is more accurate for DNA and RNA quantification and eliminates the need for time-consuming post-PCR procedures. The amount of beginning target sample and the amount of PCR product at each given cycle number have a quantifiable connection in theory. Thus, by following the fluorescence signal during cycling, one can accurately determine the quantity of starting material and product. In practice, due to non-ideal conditions that may occur during amplification (e.g., contamination, primer-dimer formation), the actual efficiency may be slightly different from what was expected based on the curve fitting. However, this will not affect the conclusion of the experiment.
In general, five fundamental PCR reagents are required for a full PCR reaction: DNA/RNA template, DNA polymerase, primers (forward and reverse), deoxynucleotide triphosphates (dNTPs), and PCR buffers.
DNA templates that can be used as sources of DNA for PCR include isolated chromosomes, purified plasmids, genomic DNA, cDNA, and RNA. Isolated chromosomes and RNA can be directly used as templates, while other DNA sources require further manipulation before use in PCR. Genomic DNA must first be converted into a more suitable form for PCR by using enzymes such as restriction endonucleases or ligases. This process is called "genomic cloning" and results in a set of DNA fragments with different lengths. Primers can then be used to amplify these fragments from the cloning vector back into the genome of the organism. The resulting set of DNA fragments contains those sequences present in the original genome that contain the same restriction sites as the cloning vector. These sites allow the fragments to be inserted back into the genome at these locations. Thus, the entire genome can be scanned for specific sequences by using multiple primer pairs that generate fragments with unique restriction sites at their ends. The amplified products from each primer pair are mixed together with the template DNA and DNA polymerase and subjected to another round of amplification.
A decent length for PCR primers is usually between 18 and 30 bases. The duration and annealing temperature are frequently related to specificity. The shorter the primers, the better they will bond or anneal to the target. The bases have an effect on the Tm as well. Melting temperatures are greater for G and C than for A and T. This is why GC-rich regions of DNA tend to be amplified first when using short primers.
Beeson's Primer (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2813200/) is a commonly used short primer that anneals to a region of DNA coding for histidine. Its use is illustrated here: https://www.ncbi.nlm.nih.gov/books/NBK171594/.
PCR requires a pair of primers that anneal to opposite strands of the target gene and have 3' ends that match the sequence of each other. The primers should be long enough to bind more than once, but not so long that they cause secondary structures in the DNA. They should also anneal at a suitable temperature. For example, if the DNA contains many A/T pairs, then it would be best to choose primers with some Gs and Cs to increase the annealing temperature. If this isn't possible, consider using a high annealing temperature to compensate.