Real-time PCR

The Polymerase chain reaction (PCR), one of the most powerful technologies in molecular biology, helps in amplifying or copying specific sequence of DNA or complementary template employing oligonucleotides, heat-stable DNA polymerase and thermal cycling. With each cycle, the number of target molecules is doubled theoretically and DNA is exponentially amplified.

Applications

  • Gene Expression (mRNA) Analysis
  • microRNA & Noncoding RNA Analysis
  • Genetic variation analysis
  • Protein Analysis
  • Mutation detection assays

PCR is the leading tool for detection and quantification of DNA and RNA. The accuracy and precision for detection within a 2-fold range

Differences: Traditional PCR vs. Real-time PCR

Traditional PCR Real-time PCR
Detection and quantification of the amplified sequence are performed at the end of last cycle. Detection and quantification of amplified sequence is carried at each cycle.
Followed by gel electrophoresis and image analysis for assessment of amplification. The amount of DNA is measured after each cycle using fluorescent dyes. The signal strength is directly proportional to the PCR product molecules.

 

 

 

 

 

STEPS

 

 

 

 

 

The following reactions occupy a central role in real-time PCR.

  1. Denaturation: Incubation at higher temperature leads to melting of dsDNA into single strands and secondary structure in single-stranded DNA is denatured. DNA polymerase can withstand temperature of 95°C and the timing is increased based on GC concentration.
  2. Annealing: Complementary sequences are presented with an opportunity to hybridize at an appropriate temperature, usually by calculating the melting point of the primers involved.
  3. Extension: The activity of DNA polymerase is optimal at 70–72°C. The extension initiated by primers can occur up to 100 bases per second. When PCR product molecules (amplicon) is small, the extension step is usually combined with annealing at 60°C temperature.

Reaction components and Parameters

DNA polymerase: The selection of DNA polymerase is critical to the success of assay. Isolated from thermophilic bacterium, the enzymes resist breaking down at higher temperatures and therefore useful in copying DNA using a polymerase chain reaction. The role of Taq polymerase is to move along the strand of DNA and use it as a pattern for assembling a new strand, complementary to the template. For as many as 75 cycles, Taq polymerase is useful in PCR to multiply DNA exponentially. Thermo stability of DNA polymerase is comprehensively examined before the selection. Hot start specific antibodies are used to block DNA polymerase from synthesizing nonspecific product resulting from mispriming. The antibody ensures DNA polymerase is not active during reaction setup and during denaturation.

Reverse transcriptase transcribes RNA into complementary DNA. Usually, the starting material is RNA. The constituent reverse transcriptase is as important as DNA polymerase as it provides high yields of full-length cDNA and active at higher temperatures. In quantification real-time PCR, reverse transcriptase active in higher temperature allows use of a gene specific primer, thereby increasing specificity and reducing background.

dNTPs (deoxynucleotide triphosphates) is a mixture of four monomeric units such as dATP, dTTP, dCTP and Dgtp. dNTP molecules contains the DNA base in a highly energized triphosphate form. These nucleotides makeup the new DNAs resulting from polymerization reaction as they are added to template strands. Each base is added to DNA through the phosphodiester bond and a molecule of pyrophosphate.

Magnesium chloride or magnesium sulfate is used at a concentration of 3 mM for most targets and the optimal concentration may vary between 3 and 6mM. Magnesium ions help increasing Taq DNA polymerase activity at the expense of specificity. Based on the assay, the concentration of magnesium used usually varies.

During replication, dntp’s gets broken down to dnmp’s to form phosphodiester bond between 3′ OH of adjacent nucleotide and 5′ Phosphate of the upcoming nucleotide. In this reaction, Mg++ binds to the alpha phosphate group of dntp and helps in the removal of beta and gamma Phosphate from dntp.

Template Use

Template nucleic acid use can range between 10 to 1000 per real-time PCR reaction. This is equivalent to cDNA generated from 1 pg to 100 ng of total RNA. Higher use of template nucleic acid may result in greater levels of contaminants that reduces PCR efficiency. Isolation of mRNA improve the yield of specific cDNAs.

Treating the template with DNase I can significantly help reduce contaminants. Intact RNA is essential for full-length, high-quality cDNA synthesis and may be important for accurate mRNA quantification.

Primer Design Guidelines

  1. Primers should be of length 18-24 nucleotides.
  2. Should be in the range of annealing temperatures.
  3. Specific for the target sequence and be free of internal secondary structure
  4. The primers should necessarily avoid stretches and repeating motifs as it can result inappropriate hybridization.
  5. Primers should have GC content of 50% and compatible melting temperatures.

Primer Design Software

Using primer design software programs ensure that primers are specific for targets and are free of secondary structure. Also, it helps in avoiding complementary hybridization at 3ʹ ends within each primer. Some of the notable primer design software programs are Invitrogen, OligoPerfect and Primer Express. These software programs are sophisticated enough to automate analysis of sequence and design primers.

Analysis

This section covers the definitions used in RT PCR analysis.

Baseline refers to the signal level during initial cycle of PCR, between cycle 3 and 15. During these cycles, there are little changes in fluorescent signal. The little changes is attributed to the background or noise of the reaction. The baseline is usually moderated to allow accuracy of the threshold cycle.

Threshold is the level of signal that reflect statistically significant rise over the calculated baseline signal. This is set to distinguish amplification signal from the background. Mostly, software sets the threshold at 10 times the standard deviation of value of the baseline.

Threshold cycle is the cycle number at which fluorescent signal crosses the threshold. The cycle number is inversely proportional to the starting amount of target. It is useful in calculating the initial DNA copy number.

The correlation coefficient is a measure of how well the data fit the standard curve.

Dynamic range is the range over which an increase in starting material concentration results in a corresponding increase in amplification product.

Advantages

  • Ability to monitor individual reaction as they happen in real time.
  • Measuring the quantity of amplicon at each cycle and thereby accurate quantification of starting material in samples.
  • Dynamic detection