PCR is highly specific and can distinguish between templates that differ in a single base only. The variable position can be interrogated using primers, probes, or post-PCR analysis of the product by high-resolution melting. What’s especially challenging is to study rare sequence variations, such as somatic mutations.
Specificity to detect one mutated sequence against a background of thousands of wild-type sequences is achieved by techniques such as castPCR, myT primers, RNaseH-dependent PCR, and SuperSelective Nucleic Acid Amplification Primers.
Genetic variation does not explain all observed hereditary phenomena; other mechanisms that do not directly depend on DNA sequences contribute. These are collectively referred to as epigenetic. They regulate cell differentiation and include DNA methylation and histone modifications.
PCR can be used to interrogate DNA methylation status either by treating with bisulfite, which converts cytosine to uracil, while 5-methyl cytosine is resistant, or by using methylation-sensitive restriction enzymes. Interaction between histones and DNA can be studied using chromatin immunoprecipitation.
Already 15 years before PCR, Howard Temin and David Baltimore independently discovered the enzyme reverse transcriptase (RT) which is a RNA-dependent DNA polymerase that uses an RNA template to produce a single-stranded DNA transcript known as complementary DNA, which then can be quantified by qPCR.
The yield of RT activity depends on template, priming strategy, and reaction conditions, and ranges between 0.5 and 80%. Performed at optimized conditions, however, RT is almost as reproducible as qPCR, allowing highly sensitive and accurate quantification of messenger RNAs and other long RNA species.
MicroRNAs are small, noncoding, single-stranded RNAs involved in posttranscriptional regulation that were discovered in 1993 by Victor Ambrose. Because they are short (22–24 bases), microRNAs are hard to quantify. Current techniques either use sequence-specific tailed RT primers to produce cDNA that is longer than the template microRNA, use thermodynamically stabilized LNA primers, or extend the microRNA first by polyadenylation or ligation.
Proteins are traditionally detected by antibodies. In 1992 Charles Cantor tagged the detection antibody in an ELISA setup with an oligo and used PCR for quantification. Because of the exponential amplification, the immuno-qPCR method had a much wider dynamic range than did ELISA.
In 2002 Ulf Landegren and his team developed proximity assays, which use pairs of antibodies tagged with oligonucleotides. When brought into proximity by binding to the same target protein, the oligonucleotides are either ligated or extended to serve as templates for qPCR.
The method is homogeneous, requiring neither support nor washing, which increases sensitivity. It is not limited to proteins; anything that can be bound by antibodies or aptamers can be quantified this way.
Other advances include the development of high-throughput platforms and preamplification that allows quantification of multiple markers in very small samples including single cells. Recently our team developed qPCR tomography to measure intracellular mRNA profiles.
A few years ago many of the leading vendor companies seemed to be spending more time developing songs for YouTube rather than novel qPCR applications and methodologies. One might have thought qPCR was fully developed.
However, the field was only taking a break. For qPCR to fully reach its potential, it must be reproducible and reliable. This is more challenging than one may think because the PCR testing process involves sampling and preanalytics, which generally contribute much greater variability to the data than the amplification itself.
Recent European studies of the proficiency of RNA analysis revealed that one-third of the laboratories had at least two quality parameters out of control. Several years back, a group of opinion leaders led by Stephen Bustin published the MIQE guidelines. These provide advice to researchers and reviewers regarding what information should be provided when qPCR data are reported.
Today many leading journals request that manuscripts be MIQE-compliant in order for them to be considered for publication. Standardizing organizations such as CEN, CLSI, and ISO have recognized this need, too. Work has been initiated to develop guidelines for the qPCR testing process, and products for quality assessment have begun to appear.
The qPCR community is becoming aware of the importance of quality control, and within the next decade or two, qPCR will be the most sensitive, specific, and also the most reliable method we have for biomolecular analysis in clinical settings.