Because significant amounts of a sample of DNA are necessary for molecular and genetic analyses, studies of isolated pieces of DNA are nearly impossible without PCR amplification.
Often heralded as one of the most important scientific advances in molecular biology, PCR revolutionized the study of DNA to such an extent that its creator, Kary B. Mullis, was awarded the Nobel Prize for Chemistry in Throughout the PCR process, DNA is subjected to repeated heating and cooling cycles during which important chemical reactions occur.
To automate this process, a machine called a thermocycler jump-starts each stage of the reaction by raising and lowering the temperature of the chemical components at specific times and for a preset number of cycles. At this point, the DNA polymerase begins making a new DNA strand by attaching to the primers and then adding dNTPs to the template strand, thereby creating a complementary copy of the target sequence Figure 4.
Figure 5: The replication cycle repeats many times. The number of new copies of the DNA sequence of interest doubles with each three-step cycle.
Thus, if the PCR process is repeated 40 or 50 times, even small samples of template DNA can yield millions of identical copies Figure 5. PCR is an incredibly versatile technique with many practical applications. Once PCR cycling is complete, the copied DNA molecules can be used for cloning, sequencing, mapping mutations, or studying gene expression. One modification of conventional PCR allows researchers to copy a particular DNA sequence and quantify it simultaneously.
This refinement involves the use of fluorescent dyes or probes that label double-stranded DNA molecules. These fluorescent markers bind to the new DNA copies as they accumulate, making "real-time" monitoring of DNA production possible. As the number of gene copies increases with each PCR cycle, the fluorescent signal becomes more intense. Plotting fluorescence against cycle number and comparing the results to a standard curve produced by real-time PCR of known amounts of DNA enables scientists to determine the amount of DNA present during each step of the PCR reaction.
This page appears in the following eBook. Aa Aa Aa. In other words, PCR enables you to produce millions of copies of a specific DNA sequence from an initially small sample — sometimes even a single copy. It is a crucial process for a range of genetic technologies and, in fact, has enabled the development of a suite of new technologies.
PCR mimics what happens in cells when DNA is copied replicated prior to cell division, but it is carried out in controlled conditions in a laboratory. The machine that is used is simply called a PCR machine or a thermocycler. Test tubes containing the DNA mixture of interest are put into the machine, and the machine changes the temperature to suit each step of the process. The separation happens by raising the temperature of the mixture, causing the hydrogen bonds between the complementary DNA strands to break.
This process is called denaturation. Primers bind to the target DNA sequences and initiate polymerisation. Add in short strings of other nucleotides, known as primers. Scientists choose a primer that will pair with — or complement — a specific series of nucleotides at the end of the DNA bit they want to find and copy. Each such series of nucleotides is known as a genetic sequence. Scientists also throw into the mix a few other ingredients, including single nucleotides, the building blocks needed to make more DNA.
Now place the test tube into a machine that heats and cools these test tubes over and over again. A normal piece of DNA is described as double-stranded. But before it prepares to reproduce itself, DNA will split down the middle of the ladder. Now the rungs separate in half, with each nucleotide remaining with its adjacent strand.
This is known as single-stranded DNA. With PCR technology, after the sample cools down again, the primers seek out and bind to the sequences they complement. Single nucleotides in the mix then pair up with the rest of the open nucleotides along the targeted single strand portion of DNA.
In this way, each original bit of target DNA becomes two new, identical ones. The primers and extra nucleotides duplicate the selected portion of DNA again.
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