Polymerase Chain Reaction (PCR) - RNA / BOC Sciences

What is Polymerase Chain Reaction?

Polymerase Chain Reaction (PCR) is a technique widely used in molecular biology, diagnostics, forensics, and molecular genetics to amplify specific regions (amplicons) of a DNA sample. PCR can amplify several molecules of a precious DNA sample to produce a large amount of DNA, ranging from 50 to more than 25,000 base pairs in length.

Fig 1. Real-time polymerase chain reaction (Yeung et al., 2006)

Steps of the Polymerase Chain Reaction

The PCR amplification process consists of three sequential steps.

Denaturation

Heat is applied to the double-stranded DNA template to dissociate it.
Annealing

Short DNA molecules called primers bind to the flanking regions of the target DNA.
Extension

The DNA polymerase extends the 3' end of the primer along the template strand. These steps are repeated ('cycled') 25-35 times to obtain an exact copy of the target DNA in an exponential manner.

Polymerase Chain Reaction Types

Quantitative Real-Time PCR (qPCR)

Quantitative real-time PCR allows for real-time monitoring of the amplification process and detection of PCR products during amplification.
Reverse Transcription PCR (RT-PCR)

Reverse transcription (RT)-PCR and RT-qPCR are two commonly used PCR variants that enable gene transcription analysis and quantification of viral RNA in clinical and research settings. RT-PCR can be performed according to two methods: one-step RT-PCR and two-step RT-PCR. In the first case, the RT reaction and the PCR reaction take place in the same tube, while in the two-step RT-PCR, the two reactions are performed in the same tube. PCR, the two reactions are separate and performed sequentially.
Reverse Transcription-quantitative PCR (RT-qPCR)

As with RT-PCR, there are two methods of quantifying RNA by RT-qPCR: one-step RT-qPCR and two-step RT-qPCR. In both cases, the RNA is first reverse-transcribed into cDNA, which serves as a template for qPCR amplification. In the two-step method, reverse transcription and qPCR amplification are performed sequentially as two separate experiments. In the one-step method, RT and qPCR are performed in the same tube.

Characteristics of PCR

High Specificity

PCR primers are designed so that the amplification reaction is highly specific, amplifying only on target sequences that exactly match the primer sequence.
High Sensitivity

PCR can amplify a large number of target sequences from a very low concentration of starting DNA or RNA template.
Simple and Fast

PCR reactions usually take only a few hours to complete and can amplify millions or even billions of copies of a target sequence in a short period.
Low Purity Requirements for Specimens

There is no need to isolate viruses or bacteria and culture cells. Both crude DNA and RNA can be used as amplification templates. DNA can be amplified directly from clinical specimens such as blood, body fluids, lavage fluids, hair, cells, biopsies, and so on.

Application of Polymerase Chain Reaction

Gene Expression

PCR can often be used to detect differences in gene expression in different cell types, tissues, and organisms at specific time points. First, RNA is isolated from the target sample, and messenger RNA (mRNA) is reverse-transcribed into complementary DNA (cDNA). Subsequently, the initial level of mRNA is determined by the amount of cDNA amplified by PCR.
Genotyping

PCR can be used to detect sequence differences in alleles in a particular cell or organism. Examples include genotyping of genetically modified organisms such as knockout and knock-in mice. Genotyping by PCR is also an essential way to genetically analyze mutations in cancer and genetic diseases.
Cloning

Since primers are synthesized in a 3' to 5' orientation, failure or insufficient synthesis of these DNA oligonucleotides will result in truncation of the 5' sequence. Therefore, it is recommended that excess synthesis reagents and non-full-length DNA oligonucleotides be removed by purification to ensure successful cloning of the target PCR fragment.
Mutation

One of the major advantages of PCR cloning is the ability to introduce the desired mutation into the target gene by cloning for mutation studies. In targeted mutagenesis, PCR primers are designed to incorporate base substitutions, deletions, or insertions into specific sequences.
Sequencing

In Sanger sequencing, PCR-amplified fragments are purified and used in the sequencing reaction. The 5' ends of PCR primers are labeled using common sequencing primer binding sites to simplify the sequencing workflow.

Frequently Asked Questions (FAQ)

What are the main types of PCR and their applications?

Key PCR types include qPCR for quantification, RT-PCR for RNA analysis, and digital PCR for absolute quantification, each serving distinct research needs in gene expression and detection.

How can PCR efficiency be optimized for challenging templates?

Optimization involves primer design, annealing temperature adjustment, magnesium concentration tuning, and polymerase selection to enhance specificity and yield.

What factors contribute to high-specificity amplification?

Primer specificity, template quality, reaction conditions, and thermal cycling parameters collectively determine amplification specificity and minimize off-target products.

How do real-time PCR systems enable quantification?

Fluorescent detection during amplification allows real-time monitoring of product accumulation, enabling precise quantification through threshold cycle (Ct) values.

What considerations are important for multiplex PCR assays?

Primer compatibility, probe spectral separation, and reaction optimization ensure simultaneous amplification of multiple targets without interference.