Structural Biochemistry/Polyermase Chain Reaction

PCR, or polymerase chain reaction, is a technique that can be used to replicate DNA by many orders of magnitude or create mutations in DNA. PCR is a valuable technique for biochemists. For example, it allows us to study the effects of genes and segments of DNA by inducing mutations. In addition, because PCR can be used to amplify even a small amount of DNA, it is a useful tool for forensic scientists. PCR amplifies DNA quickly by thermal cycling. With a thermal cycler, temperatures can be changed constantly through many cycles. This allows a thermal cycler to denature double stranded DNA, anneal primers to it, and then activate a DNA polymerase in order to replicate the DNA. By repeating this process multiple times, DNA is replicated exponentially, allowing us to study it further.

A PCR machine/Thermal Cycler

The PCR amplification of DNA undergoes three stages:

1. DNA Denaturation: The two DNA strands are separated by heat (94^oC).

2. Oligonucleotide Annealing: Two complementary oligonucleotides (primers) are added to each flanking sequence as the heated DNA strands are cooled (50-60^oC).

3. DNA Polymerization: Heat-resistant DNA polymerase catalyzes 5'-3' DNA synthesis (70^oC).

After amplification by PCR, the gene of interest is cleaved by restriction endonucleases and ligated to the plasmid cloning vector. The recombinant plasmid is subsequently placed in a bacterial host and propagated.

In addition to DNA amplification, PCR can create mutations and deletions in DNA as well as introduce a restriction endonuclease site.

History

Schematic drawing of the first four PCR cycles:
1)Denaturing at 96°C.
2)Annealing at 68°C.
3)Elongation at 72°C (P=Polymerase).

The first cycle is complete. The two resulting DNA strands make up the template DNA for the next cycle, thus doubling the amount of DNA duplicated for each new cycle.

1. Denaturation

DNA denaturation means to heat a double stranded DNA and form two separate single strands.

Basically, this process breaks the hydrogen bonds between the double helix bases in order to overcome the energy that keeps the bases so well stacked together. Many denaturation techniques exist and are possible. The most popular and used technique is by simply raising the temperature of the DNA above its melting point, Tm. The base pairs are then unstacked and can be monitored spectrophotometrically. DNA is absorbed very strongly at 260 nm, and as DNA keeps on melting, its absorbance increases because single strands are absorbed at higher wavelengths and then it stays constant once fully separated. This process is called Hypothermic Effect. This whole technique can be reversed and therefore DNA can be renatured at a certain amount of time allowing the estimation of base composition according to the time. The main biological reasons for denaturing DNA is for replication and transcription.

2. Annealing

Separated complementary strands of nucleic acids spontaneously reassociate to form a double helix when the temperature is lowered below Tm. This renaturation process is called annealing. For PCR reactions, the usual annealing time is 30-45 seconds. Increasing this time by few more minutes does not affect the results of the PCR. On the other hand, as the polymerase has some reduced activity between 45 and 65o C (temperature interval for most annealing processes), longer annealing times may increase the likelihood of unspecific amplification products. Annealing temperature is one of the most important parameters that need adjustment in the PCR reaction. Additionally, the flexibility of this parameter allows optimization of the reaction in the presence of variable amounts of other ingredients, especially template DNA. Annealing temperature is important in finding and documenting polymorphisms. Slight mismatches, (even 1 base-pair mutations) in one of sequences bound by the two primers used to amplify a DNA locus, can be detected by slight variations in annealing temperature and/or by multiplex PCR.

3. Polymerization

Polymerization is an endemic process which means energy input is required to achieve it. Also required joining of the sugar-phosphates, in addition to the nucleotides. Finally, it requires an enzyme, known as DNA polymerase. Triphosphate nucleotides make the polymerization process possible. These triphosphate nucleotides float freely within the nucleus of the cell, and each DNA base exists in a triphosphate nucleotide form. The energy that is released by the breaking of the triphosphate bond is what provides the energy for the polymerization of DNA.

PCR was an idea conceived by Kary Mullis in 1983 who, at that time, was an employee at Cetus Corporation in Berkeley, California. According to Mullis, he got the idea of this technique of DNA amplification while driving his car. Cetus Corporation saw great potential in this method and took Mullis and other scientists off their work to solely concentrate on perfecting PCR.

These scientists spent a year working on PCR, but they encountered numerous obstacles. One in particular was the use of DNA polymerase which was originally obtained from E. coli. However, PCR required heating to denature the DNA strands but this heating, in addition, would inactivate the DNA polymerase. Therefore, for each run DNA polymerase had to be added which, in total, took a lot of DNA polymerase to run the entire polymerase chain reaction. However, the discovery of Taq polymerase, which was purified and isolated from the bacterium Thermus aquaticus, led to dramatic improvements in the PCR technique since Taq polymerase is stable under high temperatures thus virtually eliminating the need to add DNA polymerase after each cycle and allowing the technique to be effectively automatized.

In 1984, the results of the first PCR were analyzed using Southern blotting and it was shown that PCR amplified the target sequence. Cloning results also proved that PCR increased this amount while still retaining high accuracy in the final results. The patent for PCR was approved in 1987. In December of 1985, Cetus started to produce commercial PCR machines and afterwards, PCR began to make its way into forensic science. In 1986, it was used by Edward Blake in the court case "Pennsylvania v. Pestinikas" and in 1989, Alec Jeffreys begins to amplify DNA from old cases thus increasing their sensitivity when tested and allowing innocent prisoners to be set free.

For his contributions to DNA-based chemistry and for the development of the PCR technique, Kary Mullis was awarded the 1993 Nobel Prize in Chemistry.

Variations of PCR

Quantitative Real Time PCR (qPCR): Quantitative PCR is a technique of PCR in which a DNA sample is simultaneously amplified and quantified. The general procedure is the same as the original PCR, however, in qPCR the sample of amplified DNA is detected in real time as the reaction progresses whereas in regular PCR, the sample is measured at the end of the reaction. Two common methods are used in qPCR which are: (1) a use of fluorescent dyes which bind to any double-stranded piece of DNA (for example, the EvaGreen dye and (2) specific fluorescent labeled probes which are detected only after hybridization with the complementary DNA strand. For both methods, an increase in measured fluorescence is proportional to an increase in DNA sample. This method of PCR is more efficient and precise than regular PCR since detection happens real time and does not require additional time running the sample on a Southern blot. There are many applications for qPCR which include, but are not limited to, the rapid diagnostic of nucleic acid diseases such as cancer and genetic abnormalities.

Reverse Transcription PCR (RT-PCR): Reverse transcription PCR is a technique of PCR to amplify, detect and quantify mRNA. PCR is first modified in the beginning by first converting RNA to a cDNA by reverse transcriptase. This part of the reaction may or may not be carried out in the same tube as PCR. The next step follows PCR by denaturing the double stranded cDNA, allowing primers to bind, and activation of the DNA polymerase to amplify the amount of DNA in the sample. Like PCR, the products of this reaction may be viewed real time, with fluorescent dyes such as SYBER Green, or the products may be assessed post-reaction. The utility of this reaction is to amplify the amount of RNA in a given sample. In addition, since the products of this reaction is the gene template for which proteins are coded from, this sequence maybe inserted into Prokaryotes for protein production, etc.

Touchdown PCR: Touchdown PCR is a modification of the original PCR technique in which the reaction is initially run at the temperature at which the annealing primers melt. In subsequent cycles, the temperature is gradually reduced, by one degrees Celsius or so, until a specific temperature, or touchdown temperature, is reached. The utility of this technique is that it allows early accumulation of specific products which later serve as templates for later PCR amplification which lowers the non-specific PCR products.

Inverse PCR: Inverse PCR is a variation of the original PCR technique in which one internal sequence of the DNA to be amplified is known. One of the limitations of the original PCR is that it requires primers complimentary to the end sequences of the DNA to be amplified. However, the utility of this technique is that if only one site is identified, then PCR may still be carried out. This is achieved by digesting the ends of the DNA by a restriction endonuclease and ligating the two free ends of the DNA with a DNA ligase to produce a circular DNA. Then the known internal sequence is cleaved with another restriction endonuclease thereby generating a strand of DNA where the end sequences are known which allows amplification of the target through PCR.

Helicase-Dependent Amplification (HDA): Helicase-Dependent Amplification is a method similar to PCR. However, HDA is different in that it relies on an enzyme helicase to split apart the DNA, rather than heat up DNA to denature it. Therefore, it is a method of PCR which operates at a constant temperature. So, the methodology is similar, the helicase enzyme separates the double-stranded DNA into single strands to which primers anneal and the DNA amplified. The utility of this technique is that it may be run almost anywhere, such as at a crime scene, as opposed to PCR which is only feasible in a laboratory setting since it requires large, expensive, and bulky machines. However, despite these positive qualities, HDA is not used as much as PCR since most of these types of reactions, whether from a crime scene or from a hospital, are run in labs most of the time. In addition, HDA cannot run as much samples as PCR and HDA is relatively more expensive than PCR.

Nested PCR: Nested PCR is a variation of PCR in which to minimize the results of primers binding to the wrong places in the DNA sequence thereby generating contamination. This reaction is first performed with a pair of primers that bind to the DNA in various places, but of importance is that they bind on the outside of the sequence of interest to be amplified. During the second cycle, a different pair of primers are used to bind to the specific sequence of DNA on the inside of the first two primers and this sequence is amplified. The reasoning behind this technique is that if a random sequence was amplified by the first primers by accident, the probability is very low that an internal sequence between the first primers would be amplified by the second primers thereby effectively minimizing contamination of side reactions.

Uses

Detecting Evolutionary Relationships: PCR can be used to amplify a small amount of DNA found in fossils. This amplified DNA can be sequenced. Using the techniques found in bioinformatics, this newly sequenced DNA can be tested for similarity with other ancient organisms as well as modern organisms. These comparisons reveal what modern species the ancient species was a common ancestor of as well as what other ancient species it was closely related to. PCR removes the restriction of bioinformatics only analyzing the DNA of modern organisms and allows distance evolutionary relationships to be more easily identified.

Detecting the Presence of Cancer Cells: If it is uncertain whether a trace of cancer cells exists in a tissue sample, the DNA can be isolated from the sample and purified. PCR can be performed on the purified DNA. The amplified DNA can more easily be tested for the presence of certain mutations (in growth genes) known to cause cancer. It isn't a foolproof way to detect cancer (some cancer cells might not have any of the list of "cancer causing mutations"), but the point is that PCR makes testing for the presence of a mutation possible when it would have been too small of a sample otherwise.

Victim's blood on suspect's shirt = GUILTY!! (probably)

Forensics: A small amount of DNA obtained from the somewhere at the crime scene (i.e. blood on a suspect's shirt) can be amplified using PCR and compared with the victim's and suspect's DNA. A fraction of a drop of blood or a single hair might not contain enough DNA for testing without PCR, but PCR makes testing this small amount of DNA possible. Sequencing the DNA is too laborious, so restriction enzymes are used to create a DNA fingerprint. The DNA sample is subjected to one or more restriction enzymes for a set length of time. The restriction enzymes cut the DNA at their recognition sequences, but not at every recognition sequence (they would if there was more time). If the DNA is different, the enzymes will cut different lengths of DNA and if the DNA is the same, the enzymes will cut the same distribution of lengths in both samples. The DNA fragments from each sample are run through a gel and compared. This can provide strong evidence of the innocence or guilt of a suspect.