Real-time PCR is a sensitive method for RNA analysis based on fluorescence measurement. It is used in basic research, applied molecular medicine, and biotechnology. Real-time PCR assays are easy to perform and combine high sensitivity with reliability. The technology is evolving rapidly with the introduction of new reagents and instrumentation. It can be used for the confirmation of data acquired by microarray analysis of gene expression. We review basic principles of real-time PCR and describe its application in hematology and especially in multiple myeloma research.
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An Overview Of Molecular Biology
DNA, the chromosomal material in the cell nucleus, is transcribed by polymerases to form RNA species with different functions. These include messenger RNA (mRNA) produced from each of the ∼20,000 protein coding genes, microRNAs (mIRs) transcribed from the ∼500 regulatory mIR genes, and ribosomal and transfer RNAs that are components of the ribosome and the protein biosynthesis machinery. mRNAs are then translated into proteins by the ribosome and then typically degraded quickly because of the actions of mIRs and cellular nucleases. The set of mRNAs and mIR genes that get transcribed in any particular cell is regulated by growth factor–responsive transcription factors, cell type–specific enhancer complexes, and the epigenetic state of the DNA surrounding genes as well as their scaffold histone proteins. Epigenetic modulation of DNA and histones occurs commonly through methylation and acetylation and is dynamically regulated during hematopoietic cell development and during the development of leukemias and lymphomas.1,2 Acquired (somatic) defects in one or more of these processes underlie the development of hematologic conditions . In addition, inherited gene defects or normal population variations in these cellular functions lead to predisposition to subsequent development of hematologic conditions.11,12 With improved understanding of the basic mechanisms underlying disease, therapies which target the type of molecular aberrations in hematologic conditions have increasingly been developed.
Figure 4.1
Polymerase chain reaction (PCR). A. A three-stage conventional PCR, with denaturation, annealing, and extension steps. Components of the typical
PCR are illustrated including a DNA template (e.g., target gene), unlabeled nucleotides (dNTPs), a DNA polymerase to copy the templates and forward (F) and reverse
(R) DNA primers, one of which is fluorescently labeled (*). B. Fluorescent products from the above PCR are then detected by capillary electrophoresis. Shown is a
trace with a normally sized 167 base pair NPM1 gene product and an abnormal copy with a 4 base pair insertion (171b) characteristic of acute myeloid leukemia.
C. Quantitative PCR using the TaqMan method with four samples showing differing amounts of the target gene as indicated by Cts ranging from 23 to 39 cycles
(arrows). A graph showing 10-fold dilutions of a reference sample is plotted below, which are used to convert Ct in patient sample into copy number. D. Design of a
TaqMan qPCR assay for detection of the JAK2 V617F mutation, with identical F and R primers but two different fluorescent probes; the red one detecting the normal
JAK2 sequence (“G” at that position), and a green probe recognizing the mutated “T” sequence. The black 3′ moiety on the probes represents the quencher dye.Polymerase Chain Reaction: The Indispensable Molecular Technique
From its first application to bacterial genetics in the early 1980s, PCR has been the central technique for amplifying genes so they can be sized to look for pathogenic insertions or deletions; sequenced to look for base pair mutations; and labeled with radioactivity, fluorochromes, or chromogenic moieties to use as probes in blots and reverse microarrays. The PCR technique involves the sequential amplification by repeated cycles of DNA denaturation, reannealing, and polymerase extension of DNA targets using flanking oligonucleotides (Fig. 4.1A). In the initial cycles of the PCR, the target is exponentially amplified before gradually plateauing when the large amount of product present tends to favor reannealing of double-stranded templates rather than primer binding/extension.
To detect the products that have been amplified by PCR, the reaction is typically run out on a solid agarose or polyacrylamide substrate or gel. These PCR amplicons can be detected by a laser using capillary electrophoresis if one of the primers has been labeled with a fluorochrome (Fig. 4.1B), or by slab gel electrophoresis followed by post-staining with a DNA-binding dye (e.g., ethidium bromide) that can be visualized with ultraviolet light (see Fig. 4.2, Step 1). As described above, if RNA is to be analyzed by PCR, it is first converted into cDNA in a technique known as RT-PCR. If fluorescent probes are added into the reaction, real-time or quantitative PCR (qPCR) can be performed to calculate the amount of an RNA or DNA target present in the initial sample. A common qPCR design is the TaqMan short, gene-specific probe that has a reporter fluorophore at its 5′ end and a quencher molecule at the 3′ end. The probe hybridizes to its target amplicon during the annealing step of each PCR cycle and is then hydrolyzed by the 5′ exonuclease activity of Taq polymerase during DNA extension. When the TaqMan probe is hydrolyzed, the reporter fluorophore is detached from the adjacent quencher molecule and fluoresces in an amount proportional to the degree of PCR product amplification. Thus, as probe is bound to template and its reporter released by the polymerase extension, the detected fluorescence rises exponentially. In qPCR, the amount of initial target present in a PCR is backcalculated by observing the PCR cycle in which the fluorescence signal first becomes detectable. This threshold cycle (Ct) can then be used for absolute or relative quantitation. For absolute quantitation, the observed Ct is converted to a target copy number by plotting it on a standard curve (log Ct vs. starting copy number) constructed from samples with a known target copy number (Fig. 4.1C). For relative quantitation, target quantities are expressed relative to a co-amplified normalizer control (e.g., a highly expressed housekeeping gene such as ACTB [b-ACTIN] or ABL1). The quantity is then represented as a relative ratio most commonly the delta-Ct calculation: [relative quantity] = 2−(Ct of gene target – Ct of reference gene). A specialized form of qPCR used to detect single base pair changes in DNA is allele-specific (AS)-PCR. This method compares the amplification levels of a PCR probe or primer that recognizes one allele versus the signal from a probe that recognizes only the other allele. This same protocol can also be used to sensitively detect the level of mutated sequences in neoplasms.19 This method can routinely detect the presence of a mutation down to 0.1% of the template in the sample (Fig. 4.1D).
DNA Sequencing: The Technique Driving the Genomic Revolution
The DNA sequence of genes is built up from combinations of four nucleotides, adenine (A), cytosine (C), guanine (G), and thymine (T), and their epigenetically modified variants, particularly 5-methylcytosine. DNA sequencing to determine the base composition of the genome was first routinely applied in the late 1970s but has remained a difficult and expensive technique until the last several years. The accurate but costly gold-standard technique for determining DNA base composition, developed by Frederic Sanger, is called the dideoxy chain termination method.20 After an initial PCR step to amplify the gene of interest, this method relies on a second asymmetric PCR step in which stops in the PCR extension are randomly introduced at each position in the product by adding fluorescently labeled chain terminating variants of the A, C, G, and T nucleotides, each terminating nucleotide being labeled with a different color (green, blue, black, and red). This range of DNA molecules each terminated at a different position are then separated by size using electrophoresis and the sequence read by laser detection of the terminally labeled nucleotide (Fig. 4.2).
DNA Sequencing.
Steps in the dideoxy chain termination (Sanger) method include: Step 1: Standard polymerase chain reaction (PCR) to produce large amounts of a genespecific template, detected by slab electrophoresis followed by ethidium bromide staining of the gel. Step 2: Unidirectional (or asymmetric) PCR using the template from the first PCR along with either a forward or reverse primer in a reaction containing normal nucleotides mixed with chain terminating A, C, G, and T bases. Step 3: The range of products from the asymmetric PCR which are terminated at every possible base in the PCR amplicon are then separated by capillary electrophoresis and detected by a laser recognizing the fluorochrome/nucleotide present at the end of products. Base-calling is performed using software which normalizes the peak heights to produce the depicted electropherogram.
Molecular Diagnostic Applications In Hematology
The diagnosis of specific types of lymphoid and myeloid malignancies is discussed elsewhere in this volume, but here we summarize generally how molecular techniques are used to assist in their diagnosis. The current schema for diagnosis of hematologic neoplasms is the World Health Organization (WHO) Classification of Hematologic and Lymphoid Neoplasms.23 This classification incorporates morphology and immunophenotypic features but also increasingly relies on molecular and cytogenetic testing for definitive diagnosis.
- Molecular Diagnostics of Myeloid Neoplasms
- Molecular Diagnostics in Lymphomas
and Benign Lymphoid ExpansionsRef: https://pdfs.semanticscholar.org/f4e1/f951bcbc27420e39550b9dfa2c8bbf237802.pdf
