Structural Biochemistry/Polyermase Chain Reaction/Uses of PCR

PCR is a useful technique for scientists because it allows for the amplification and mutation of DNA. Through PCR, small quantities of DNA can be replicated by orders of magnitude, not only essentially preserving the sample if successful, but allowing for study on a much larger scale. Without PCR, the samples we could analyze and mutations we could induce and study would be limited by the amount of DNA we had to work with. Through PCR, that DNA is essentially limitless, allowing biochemists to induce various mutations in different genes for further study.

Through site-directed mutagenesis or customized primers, individual mutations in DNA can be made. By changing the amino acids transcribed from DNA through individual mutations, the importance of those amino acids with respect to gene function can be analyzed. However, this process can be difficult, particularly when genes act in concert (with varying expression with respect to gene activity). The length of time it takes to run a successful PCR and perform other techniques before study can be done (protein expression, isolation, and purification, for example), makes biochemical research time-consuming and difficult. However, PCR, coupled with other biochemical techniques, allows us to analyze the very core of organisms and the processes by which they function.

PCR is a reliable method to detect all mutations associated with genetic diseases, starting from insertion to deletions and much more. It can also be used to detect the presence of unwanted genetic materials, like infections and bacteria. For example, in diagnosis of diseases like AIDS, PCR can be used to detect the small percentage of cells that infected HIV. PCR also allows early diagnosis of unwanted diseases such as leukemia. PCR offers very simple and fast alternative.

PCR examples in research

PCR run by Nisha Patel, UCSD Pediatrics, Sander Lab

PCR is a highly, commonly used tool in research that allows researchers to amplify DNA segments with simple reactions that can be set up and finished within an hour. PCR in research is most used as a test to see if a gene is expressed in vivo. Such examples include PCR of mouse DNA in order to determine what genes that mouse carries. These mice, with specific target genes, can be studied to see the biological effects of such genes. Common PCR protocols in labs today include knockout genotyping, fluorescence genotyping, and mutant genotyping. Researchers can use PCR as a method of searching for genes by using primers that flank the target sequence of the gene along with all other necessary components for PCR. If the gene is present, the primers will bind and amplify the DNA, giving a band of amplified DNA on the agarose gel that will be run. If the gene is not present, the primers will not anneal and no amplification will occur. Amplification can occur in more that one spot in the DNA, that is why researchers can use DNA ladders to compare the relative length of the amplified strand (usually in bp). If the length of the gene is known, then researchers will know which bands to look for and which to possibly ignore. The term possibly ignore is used because PCRs are usually run with the DNA in question as well as controls, which can include DNA positive for the gene, DNA negative for the gene, and only reactants in the PCR reaction other than DNA. If there are bands positive for the gene in both the DNA negative and the only reactants reactions, as well as all other reactions, it is understood that some of the reactants are contaminated with positive DNA and should thus be thrown out and the PCR run again.

This is an example of a gel run using PCR reaction products. The columns on the far left are 100bp ladders which track the movement of segments of 100, 200, 300, etc. base pairs. Each of the wells represents the DNA taken from a mouse. As seen in the picture, some mice lack one of the DNA sequences that would be amplified by use of the primers. Here it is seen how strong of a tool PCR can be in identifying the targeted sequences of DNA in respect to mice which carry those genes. Since mice of different genetic backgrounds can be distinguished, further conclusions about certain genes can be made.

PCR Applications

PCR can be used to isolate genomic DNA. This means that PCR can isolate DNA fragments from genomic DNA by amplifying a specific region of the parent DNA strand. Applications of PCR include generating hybridization probes for Southern and Northern Hybridization, DNA cloning, DNA sequencing, genetic fingerprinting, etc.

Forensics An individual DNA profile is highly distinctive. PCR amplification of multiple genes is being used to establish biological parentage in paternity cases. Analyses of blood stains and semen samples by PCR have implicated guilt or innocence in numerous assault and rape cases. The root of a single shed hair or skin found at a crime scene contains enough DNA for typing by PCR. The power of PCR lies in how little DNA strands are required for the process to occur since amplification occurs exponentially. PCR also does not require knowledge of the targeted DNA sequence, rather it only requires the knowledge of the flanking DNA sequence.

Evolutionary Large fragments of DNA can remain intact for thousands of years due to DNA's stability. Thus, PCR is ideal for amplifying ancient DNA molecules for evolutionary studies. PCR can also be used to amplify DNA from microorganisms that have not yet been isolated.

PCR Amplication

PCR can analyze extremely small amounts of DNA sample and can amplify targeted regions of the DNA.

Quantification PCR

Quantification PCR is a method that can estimate the amount of sequence will be shown in a sample; this is normally applied to quantitatively to determine levels of gene expression. Real-time PCR is a tool for DNA quantification that measures the DNA product after each PCR amplification cycle.

PCR to diagnose diseases

PCR diagnosis of malignant diseases (i.e. leukemia) are being used routinely in cancer research. PCR assays can detect specific malignant cells at higher folds than any other methods when performing directly on the genomic DNA. This technique can identify mutations of certain growth control genes such as the ras gene. The capacity to greatly amplify selected regions of DNA can also be highly informative in monitoring cancer chemotherapy. PCR tests can detect when chancerous cells have been eliminated as well as detect a relapse.

PCR can reveal the presence of human immunodeficiency virus in people who have not mounted an immune response to this pathogen (which would otherwise be missed with an antibody assay).

Finding Mycobacterium tuberculosis bacilli in tissue specimens is slow and laborious, but with PCR as few as 10 tubercle bacilli per million human cells can be readily detected.