Conventional PCR, One-step RT PCR or Two-step real-time RT PCR: Which one is the best option?

Recombinant DNA Technology Process Polymerase chain reaction PCR

The Polymerase chain reaction (PCR) is a lab technique used for the amplification of a particular DNA or RNA fragment at a very simple enzyme response. Compared to traditional methods of DNA cloning and amplification, which can often take days, PCR requires only a few hours. PCR is highly sensitive and requires minimal template for detection and amplification of specific sequences. The fundamental PCR method was altered to expand its application. Another popular variant is reverse transcription polymerase chain reaction (RT-PCR), a method used to discover and quantify RNA. PCR technology has altered the area of molecular biology and clinical research. Due to its widespread usage, it’s very important to comprehend the scientific principles of PCR. There is a brief explanation of the different methods: Conventional PCR, RT-PCR, qPCR, and RT-qPCR.

Conventional PCR

PCR components needed to perform conventional PCR
Conventional PCR

The PCR mechanism is as straightforward as its function:

1) double-stranded DNA (dsDNA) is heat denatured

2) primers align into one DNA strands

3) the primers are extended by DNA polymerase, resulting in 2 copies of the original DNA strand. The denaturation, annealing, and elongation procedure above a collection of times and temperatures is called a single cycle of amplification. Each step of this cycle ought to be optimized to your primer and template set used. This cycle has been repeated about 20-40 times along with the amplified product can then be examined. PCR is widely utilized to amplify DNA for following experimental usage. PCR also includes applications in genetic testing or to the discovery of pathogenic DNA. In order to perform it, you need DNA polymerase, magnesium, nucleotides, primers, the DNA template to be amplified and a thermocycler.

Since PCR is an extremely sensitive system and very tiny quantities are needed for single responses, preparation of a master mix for many reactions is preferred. The master mix has to be well blended and then divide by the amount of reactions, ensuring that every reaction will include exactly the exact same quantity of enzyme, dNTPs and primers. Many manufacturers provide PCR combinations that currently include all except primers and the DNA template.

Guanine/Cytosine-rich (GC-rich) regions represent a struggle in conventional PCR methods. GC-rich strings are somewhat more secure compared to sequences with lower GC content. Because of this, GC-rich double strands are hard to fully separate throughout the denaturation stage. Thus, DNA polymerase can’t synthesize the strand without any hindrance. Added reagents may boost the amplification of all GC-rich sequences. DMSO, glycerol and betting help disrupt the secondary structures which are brought on by GC interactions and thus ease separation of the dual strands.

What is Hot Start PCR?

Difference between Conventional and Hot Start PCR
Hot Start PCR

The unspecific amplification is an issue that could happen during PCR. Most DNA polymerases perform the best at 68 – 72°C.As a result, the extension temperature ought to be in this range. The enzyme can, however, also be active to some lesser degree, in reduced temperatures. At temperatures which are much below the annealing Temperature, primers have a tendency to bind non-specifically, which may result inon-specific amplification even though the reaction was set on ice. This Can be avoided by employing polymerase inhibitors which dissociate in the DNA polymerase just once a particular temperature is reached. The inhibitor could be an antibody that connects with the polymerase and denatures the first denaturation temperature.

What is RT-PCR?

Reverse transcription PCR, or also known as RT-PCR, allows the use of RNA as a template. An extra measure permits the detection and amplification of RNA.The RNA is reverse transcribed into complementary DNA (cDNA), using reverse transcriptase. The quality and purity of the RNA template are vital for the achievement of RT-PCR. The very first step of RT-PCR is that the synthesis of a DNA/RNA hybrid.Reverse transcriptase also features an RNase H function, which degrades the RNA part of the hybrid.The single stranded DNA molecule is subsequently finished by the DNA-dependent DNA polymerase activity of the reverse transcriptase to cDNA. The efficacy of this first-strand reaction can influence the amplification procedure.From here, the conventional PCR process is utilized to amplify the cDNA. The chance to revert RNA to cDNA from RT-PCR has many benefits. RNA is single-stranded and incredibly unstable, making it tough to use. Most often, it functions as a very first step in qPCR, which quantifies RNA transcripts in a biological sample.

What is Real-Time RT PCR?

RT-PCR can be achieved in a one-step assay mixing Reverse Transcription and PCR in one reaction chamber or two-steps separating the two procedures in various reaction chambers.
RT-PCR: one and two steps processes

Real-Time PCR has become an increasingly common technique for evaluation of gene expression. There are two key techniques of real time PCR which may be carried out. The first entails such as the reverse transcriptase step in precisely the exact same tubing as the PCR reaction (one-step). The second procedure involves producing cDNA first by way of another reverse transcription reaction and then incorporating the cDNA into the PCR reaction (two-step). There are benefits and disadvantages to the two systems which you ought to consider prior to picking the best one for your experiment, such as the ease of use and price of response to the consequent yield and arrangement representation. One-step reactions are definitely easier to install with less complete hands-on time, but don’t offer the flexibility and management which are potential with two-step reactions.

One-step real-time RT-PCR

  • Easy and quick
  • Fewer pipetting measures (reducing potential errors and Contamination )
  • Finest Alternative for high-throughput screening
  • Finest way when just a couple assays are conducted
  • Accurate representation of target copy number
  • Multiplex PCR of both genes of control and interest could be Performed in single nicely, from the same RNA sample

Gene-specific primers are used for producing the cDNA and also for following amplification in 1 tube reducing experimental version because both enzymatic reactions occur under exactly the very same conditions, making one-step real-time RT-PCR highly reproducible. There are numerous different benefits of one-step reactions, such as comprise restricted sample handling and decreased seat time, which will help to reduce chances for pipetting errors and cross contamination between RT and real time PCR measures. This approach is fast to establish and also makes processing multiple RNA samples simple (particularly when utilizing liquid handling robotics), even once you’re amplifying just a few genes of interest. It’s thus ideal for high throughput screening labs where just a couple assays are run differently, with well-established response conditions, with the extra benefit that multiplex PCR of the receptor of interest and management genes may be completed in only nicely, from the same RNA sample.

What should be considered before choosing this PCR technique?

  • Normally less sensitive as It’s impossible to Maximize Both reactions Individually
  • Hard to troubleshoot RT Measure
  • No Inventory of cDNA

Response is essential, as each one the cDNA is employed for the following PCR measure, also the response conditions required to encourage both RT and PCR might not be ideal for either response, influence yield and efficiency. As a result of this, one-step reactions might require considerably more RNA into your primary samples if you’re performing many amplifications and variant between these RT reactions may complicate assay translation significantly. One-step real time RT-PCR is thus normally less sensitive compared to two-step RT-PCR.

One-step real time RT-PCR also needs careful evaluation to stop Primer dimer formation since the primers will be found throughout the decrease temperature states of the RT reaction in addition to the PCR cycling.

Two-step real-time RT-PCR

  • 2 buffers optimized for separate RT and real time PCR
  • exceptionally sensitive
  • Potentially more effective since arbitrary primers and oligo d(T) may be utilized
  • Possibility to inventory cDNA to measure several goals
  • Recommended while the reaction is done using a limiting amount of starting material.

What should be considered before choosing this PCR technique?

  • Time-consuming
  •     More speps when pipetting (increases potential error and contamination)
  •     Requires Additional optimization

With two-step real time PCR, the usage of several tubes signifies it Is more time consuming and not as flexible to fluid handling robotics and so more challenging to embrace for high throughput screening assays. The usage of several tubes and pipetting measures also exposes the response into some greater risk of DNA contamination

What is qPCR and RT-qPCR?

Quantitative PCR (qPCR) can be utilized to discover, characterize and quantify nucleic acids for many uses. Ordinarily, in RT-qPCR, RNA transcripts are measured by reverse transcribing them to cDNA very first, as explained above and qPCR is then completed. In the conventional PCR, DNA is amplified by 3 repeating steps: denaturation, annealing and elongation.Nonetheless, in qPCR, fluorescent tagging allows the selection of information as PCR progresses. This technique has many advantages because of a selection of approaches and chemistries available.

In dye-based qPCR (generally green), fluorescent labeling permits the quantification of the amplified DNA molecules by utilizing the use of a dsDNA binding dye. The fluorescence signal rises proportionally to the sum of repeated DNA and therefore the DNA is measured in“real time”;. The downsides of dye-based qPCR are that only 1 target can be analyzed at a time and the dye will seep to some ds-DNA within the sample.

In probe-based qPCR, many goals are available simultaneously in every sample but this necessitates design and optimization of a goal particular probe(s), utilized along with primers. There are lots of varieties of probe designs accessible, however, the most frequent form is a hydrolysis probe, which comprises the usage of a fluorophore and quencher. Fluorescence resonance energy transfer (FRET) prevents the emission of this fluorophore by the quencher whereas the probe is intact. During the PCR reaction, the probe is hydrolyzed during primer extension and amplification of the specific sequence it is bound to. The cleavage of the probe separates the fluorophore from the quencher also ends in an amplification-dependent growth in fluorescence. Therefore, the fluorescence signal in the probe-based qPCR response is proportional to the quantity of the probe target sequence within the sample. Since probe-based qPCR is much more special than dye-based qPCR, it’s frequently the technology employed in qPCR diagnostic assays.