Two-step Polymerase Chain Reaction (PCR) is a molecular biology technique used to analyze ribonucleic acid (RNA). It converts RNA into deoxyribonucleic acid (DNA) for subsequent amplification of specific DNA segments. As a variation of standard PCR, it allows for studying gene expression and detecting RNA-based pathogens.
What is PCR?
Polymerase Chain Reaction, or PCR, is a laboratory technique used to create millions of copies of a specific DNA segment from a small initial sample. It operates on the principle of enzymatic replication of DNA. The process involves a series of rapid temperature changes, known as thermal cycling, performed by a machine called a thermal cycler.
Each cycle of PCR consists of three main steps. First, denaturation involves heating the double-stranded DNA to 94-98°C, which separates the DNA into two single strands. Second, annealing occurs when the temperature is lowered to around 54-60°C, allowing short DNA sequences called primers to bind to their complementary regions on the single-stranded DNA templates. Finally, extension takes place at an optimal temperature, typically 72°C, where a heat-stable enzyme, Taq polymerase, adds DNA building blocks (nucleotides) to the primers, synthesizing new complementary DNA strands. These cycles are repeated 20-40 times, doubling the number of DNA copies in each cycle, leading to exponential amplification of the target DNA.
The Two Steps Explained
Two-step PCR, also known as reverse transcription PCR (RT-PCR), involves two distinct reactions performed sequentially. This method is necessary because standard PCR amplifies DNA, but many biological studies require RNA analysis.
The first step is reverse transcription, where RNA is converted into complementary DNA (cDNA). This conversion is catalyzed by an enzyme called reverse transcriptase, which uses the RNA molecule as a template to synthesize a single-stranded DNA copy. Various types of primers can be used to initiate this process, including oligo(dT) primers that bind to the poly-A tail of messenger RNA (mRNA), random hexamers that bind throughout the RNA, or gene-specific primers targeting a particular RNA sequence. The resulting cDNA is more stable than RNA and serves as the template for subsequent amplification.
The second step involves using the newly synthesized cDNA as the template for standard PCR amplification. An aliquot of the cDNA from the reverse transcription reaction is transferred to a new tube containing PCR reagents, including DNA polymerase, primers specific to the target sequence, and nucleotides. The PCR then proceeds through denaturation, annealing, and extension cycles, exponentially amplifying the specific DNA segment copied from the original RNA. This sequential approach provides flexibility in optimizing reaction conditions for both the reverse transcription and PCR steps, and allows for cDNA storage for future analyses.
Where Two-Step PCR is Applied
Two-step PCR is used in molecular biology for various applications, particularly when the starting material is RNA. A primary application is in studying gene expression, where scientists quantify the levels of specific RNA transcripts in a sample. By converting mRNA into cDNA, researchers can determine how actively certain genes are being expressed within cells or tissues. This method allows for accurate RNA quantification, even from limited sample quantities.
Another application is the detection and quantification of RNA viruses, such as Human Immunodeficiency Virus (HIV) or SARS-CoV-2. Since these viruses have RNA genomes, two-step PCR provides a sensitive method to identify their presence in clinical samples. The technique’s ability to first convert viral RNA into DNA makes it suitable for diagnostic testing and monitoring viral loads.
Two-step PCR is also beneficial for analyzing rare RNA transcripts or when multiple genes need to be studied from a single RNA sample. The generation of a stable cDNA pool during the reverse transcription step allows for multiple PCR amplifications from the same initial RNA sample. This flexibility in primer selection and the ability to optimize each reaction separately make two-step PCR an effective method for complex RNA analysis, offering higher resolution and greater control compared to one-step methods.