Which of the following is not one of the three steps of the polymerase chain reaction (pcr)?

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PCR is shorthand for a simple but very useful procedure in molecular biology called the polymerase chain reaction. It is a technique used to amplify a segment of DNA of interest or produce lots and lots of copies. 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.

How does PCR work?

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.

Standard ingredients in the mixture are:

• the DNA segment of interest
• specific primers
• heat-resistant DNA polymerase enzyme
• the four different types of DNA nucleotides
• the salts needed to create a suitable environment for the enzyme to act.

What is the PCR process?

Step 1: Denaturation

As in DNA replication, the two strands in the DNA double helix need to be separated.

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.

Step 2: Annealing

Primers bind to the target DNA sequences and initiate polymerisation. This can only occur once the temperature of the solution has been lowered. One primer binds to each strand.

Step 3: Extension

New strands of DNA are made using the original strands as templates. A DNA polymerase enzyme joins free DNA nucleotides together. This enzyme is often Taq polymerase, an enzyme originally isolated from a thermophilic bacteria called Thermus aquaticus. The order in which the free nucleotides are added is determined by the sequence of nucleotides in the original (template) DNA strand.

The result of one cycle of PCR is two double-stranded sequences of target DNA, each containing one newly made strand and one original strand.

The cycle is repeated many times (usually 20–30) as most processes using PCR need large quantities of DNA. It only takes 2–3 hours to get a billion or so copies.

PCR technology is still developing. There is continuing development and refinement of the processes and tools used, allowing the process to be adapted to meet specialist needs. For instance, new methods and refinements are being developed and used, especially when quantification of DNA in a sample is needed. New methods include real-time PCR or quantitative PCR (qPCR) and digital PCR (dPCR). In qPCR, the amplification of DNA is monitored in real time, allowing the quantification of target DNA throughout the process. dPCR is a new, more refined approach that breaks the PCR process up into many smaller steps. It offers increased precision, more reliable measurements and absolute quantification from very small or mixed samples.

Nature of science

The development of new technologies, like PCR, enables new discoveries to be made. Other technologies can also be developed. See What is PCR used for? for some examples of how PCR is used and the different types of investigations and processes that are possible because of it.

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The polymerase chain reaction, or PCR, is one of the most well-known techniques in molecular biology. Replication of single-stranded DNA from a template using synthetic primers and a DNA polymerase was first reported as early as the 1970s [1,2]. Nevertheless, the PCR method as we know it today to amplify target DNA was not developed as a research tool until 1983, by Kary Mullis [3,4]. Since then, PCR has become an integral part of molecular biology, with applications ranging from basic research to disease diagnostics, agricultural testing, and forensic investigation. For his invention, Kary Mullis was awarded the Nobel Prize in Chemistry in 1993.

PCR is a biochemical process capable of amplifying a single DNA molecule into millions of copies in a short time. Amplification is achieved by a series of three steps: (1) denaturation, in which double-stranded DNA templates are heated to separate the strands; (2) annealing, in which short DNA molecules called primers bind to flanking regions of the target DNA; and (3) extension, in which DNA polymerase extends the 3′ end of each primer along the template strands. These steps are repeated (“cycled”) 25–35 times to exponentially produce exact copies of the target DNA (Figure 1).

Over the years, the fundamental principles of PCR have remained the same, but methods have evolved with vast performance improvements to DNA polymerases and reagents, as well as innovations in instrumentation and the plastic vessels that hold the reactions.

Figure 1. Three steps of PCR─denaturation, annealing, and extension─as shown in the first cycle, and the exponential amplification of target DNA with repeated cycling.
 

DNA polymerases are critical components in PCR, since they synthesize the new complementary strands from the single-stranded DNA templates. All DNA polymerases possess 5′→ 3′ polymerase activity, which is the incorporation of nucleotides to extend primers at their 3′ ends in the 5’ to 3’ direction (Figure 2).

In the early days of PCR, the Klenow fragment of DNA polymerase I from E. coli was used to generate the new daughter strands [3]. However, this E. coli enzyme is heat-sensitive and easily destroyed at the high denaturing temperatures that precede the annealing and extension steps. Thus, the enzyme needed to be replenished at the annealing step of each cycle throughout the process.

The discovery of thermostable DNA polymerases proved to be an important advancement, opening tremendous opportunities for the improvement of PCR methods by enabling longer-term stability of the reactions. One of the best-known thermostable DNA polymerases is Taq DNA polymerase, isolated from the thermophilic bacterial species Thermus aquaticus in 1976 [5,6]. In the first report in 1988 [7], researchers demonstrated Taq DNA polymerase’s retention of activity above 75°C, making continuous cycling without manual addition of fresh enzyme possible, and thus enabling workflow automation. Furthermore, compared to E. coli DNA polymerase, Taq DNA polymerase produced longer PCR amplicons with higher sensitivity, specificity, and yield. For all the aforementioned reasons, Taq DNA polymerase was named “Molecule of the Year” by the journal Science in 1989 [8].

Figure 2. DNA polymerase extending the 3′ end of a PCR primer in the 5′ to 3′ direction.

Although Taq DNA polymerase significantly improved PCR protocols, the enzyme still presented some drawbacks. Taq DNA polymerase is relatively unstable above 90°C during denaturation of DNA strands. This is especially problematic for DNA templates with high GC content and/or strong secondary structures that require higher temperatures for separation. The enzyme also lacks proofreading activity; therefore, Taq DNA polymerase can misincorporate nucleotides during amplification. Where sequence accuracy is critical, PCR amplicons with errors are not desirable for cloning and sequencing. In addition, the error-prone nature of Taq DNA polymerase contributes to its inability to amplify fragments longer than 5 kb in general. To overcome such shortcomings, better-performing DNA polymerases are continually being developed to harness the power of PCR across a variety of biological applications (learn more about DNA polymerase characteristics).

What are the 3 steps of PCR?

What are the 3 steps in PCR cycle with its temperature?

What are the three steps of PCR quizlet?

Which of the following is not a use of polymerase chain reaction PCR?