Polymerase chain reaction (PCR) is a process that
produces many copies of the nucleic-acid sequence of interest for the
purpose of detection and/or quantification (for example, a genetic
mutation associated with a disease such as cystic fibrosis, or a foreign
agent such as a virus or a bacteria). The process involves cycling at
least two different temperatures several times to make millions of
copies of the nucleic-acid sequence of interest. One cycle includes a
temperature for separating double-stranded nucleic acid into single
strands, and a second temperature for annealing of sequence-specific
primers and for extension of the copy using the primers as a starting
point.
The enzyme, called a DNA polymerase, makes the copy
strand. Normally, after the PCR process, gel electrophoresis is used to
detect the PCR products. The addition of fluorescent probes to the PCR
process, however, allows for real-time detection, thus eliminating the
need for gel electrophoresis.
Real-time PCR has rapidly become a standard method
for clinical molecular biology laboratories because of its ability to
provide sensitive and highly specific results. Sensitivity, automation,
and turnaround time have made real-time PCR particularly useful for
pathogen detection, especially for organisms difficult to grow in
culture.
Strain variability of genetic sequences from
pathogens, however, complicates probe and primer design, and
interpretation of results. While designers typically target regions that
are conserved across various strains, these regions can often be too
short or problematic for traditional real-time PCR chemistries.
Additionally, variants with unknown polymorphisms in these conserved
regions may escape detection. The minor groove binder (MGB) probe
technology overcomes some of these obstacles.
The MGB probe technology utilizes a minor groove
binder attached to single-stranded DNA probes to boost the stability and
raise the melting temperature (Tm) of the DNA duplex formed when probes
bind to complementary targets. This enables the use of shorter probes
capable of detecting short conserved regions when developing assays to
detect multiple strains. 5′-MGB probe allows post-amplification
melt-curve analysis confirming real-time amplification results.
MGB — the minor groove binder
Minor groove binders are a potent class of naturally occurring antibiotics that bind to duplex DNA specifically in the minor groove.
Minor groove binders are a potent class of naturally
occurring antibiotics that bind to duplex DNA specifically in the minor
groove. Minor groove binders are long, flat molecules composed of
several similar subunits that are held together by peptide bonds that
can adopt a “crescent” shape. This shape allows the MGB moiety to fit
snugly into the minor groove — the deep narrow space between the two
phosphate-sugar backbones in the double helix.
The MGB moiety is stabilized in the minor groove by
hydrophobic interactions. By attaching the MGB moiety to the 5′-end,
which is shown in the graphic here, of a DNA probe during synthesis on a
commercial synthesizer or post-synthetically to an amine-modified oligo,
the MGB moiety folds back into the minor groove and stabilizes the DNA
probe-target duplex. The effect of this stabilization is an increase in
melting temperature, allowing the use of shorter probes, which can
improve mismatch discrimination.
Prior to the introduction of MGB technology,
researchers typically needed to increase the size of the probe in order
to produce melting temperatures consistent with efficient PCR. Longer
probes reduce design flexibility when restricted by small target regions
and are less sensitive to mismatch discrimination.
Another key differentiating feature of the 5′-MGB
real-time PCR technology used by Florida Hospital’s Molecular
Diagnostics Laboratory is the post-PCR melt-curve capability. With this
cleaved probe, users do not have the added confidence of the melt-curve
confirmation. The 5′-labeled MGB probes are resistant to the polymerase
exonuclease activity. The probes remain intact after amplification and
that allows the possibility of doing confirmatory melt curves and
detecting unknown mutations.
Universal cycling
In the Florida Hospital’s Molecular Diagnostics
Laboratory, cytomegalovirus (CMV), Epstein-Barr virus
(EBV), and BK
virus (BK) tests were developed using the real-time PCR reagents from
the company that provides the lab’s MGB probe technology. With the MGB
real-time PCR technology, the laboratory is able to perform all three
tests (BK, CMV, and EBV), if needed, from one sample extraction since it
has optimized the three tests to use the same internal control
templates. All that is needed is to extract one specimen to place in all
three PCR master mixes. This certainly saves a lot of time.
Another key benefit of the MGB real-time PCR
technology is that all reagents run under the same cycling condition —
universal cycling condition. This reduces the chance of errors in the
lab because one cycling condition is used for every test.
When bringing in new tests, the set up is simple. The
optimal PCR cycling condition is already known; all that needs to be
done is to validate the specimen types and extraction protocol, and
analyze the results for the new test.
The MGB real-time PCR technology also allows multiple
tests to be run on the same thermal cycler. This enables labs to run
low-volume tests without waiting for enough samples for a batch, which
greatly improves lab efficiency and turnaround time.
Summary
The advantages of MGB real-time PCR technology are:
- use of short, or highly conserved, specific sequence;
- specific primers and non-cleavable probes;
- post-PCR melt-curve analysis;
- universal cycling conditions;
- open platform; compatible with most real-time PCR instruments;
- unsurpassed sensitivity;
- multiplexing with a rich proprietary dye set; and
- the ability to tailor designs to detect most mutations.
George Corpus, CLSp(MB), is the manager of the
Molecular Diagnostics Laboratory at Florida Hospital in Orlando, FL,
which uses TaqMan Roche Molecular Systems and MGB Alert from Wescor’s
Epoch Biosciences.
