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High-fidelity DNA polymerases mediate geno-typing
3´Terminal-labeled primer extension Terminal-labeled
primer extension is a single nucleotide polymorphism (SNP) assay
consisting of 3´ terminal-labeled, allele-specific primers
and DNA polymerases with proofreading activity. Both 3´ terminal
[ 3H]-labeled and fluorescent-labeled primers have been
successfully applied in genotyping analyses. The 3´ terminal
mismatched nucleotide that bears the signal to be detected was removed
by the proofreading function, whereas the label was retained when
the primer and template were perfectly matched. The terminal-labeled
primer extension approach has several advantages over current SNP
assays. The most significant advantage is that it greatly decreases
false positive results by a direct consequence of the proofreading
activity of Exo+ polymerases. The second advantage of
terminal-labeled primer extension is its high sensitivity. Terminally
labeled primer extension harnesses the power of polymerase chain
reaction (PCR) to improve the efficiency of genetic analysis[1-7].
SNP-triggered on/off switch Exo+ polymerases
together with 3´ phosphorothioate-modified mismatched primers
work as an off switch in DNA polymerization. Phosphorothioate modification
renders oligonucleotides nuclease-resistant, which blocks mismatch
excision, a strategy widely used in antisense technology as well
as in single base extension. For 3´ allele-specific primers
with phosphorothioate modification, a perfectly matched primer turns
on DNA polymerization, whereas mismatched primers turn it off, resulting
in no product. This breakthrough observation of an on/off switch
action has been repeatedly confirmed using either short artificial
amplicons or natural genomic DNA templates. The off switch directly
resulted from 3´exonuclease activity and has been well supported
by comparisons of a variety of DNA polymerases in both linear and
exponential amplification with phosphorothioate-modified, allele-specific
primers[3-13].
The crucial structural components of the on/off switch are: (i)
allele-specific primers with 3´ terminal exonuclease-resistant
modification; and (ii) DNA polymerases possessing 3´ exonuclease
activity. In recent studies by Di Giusto et al[6,7,14],
four types of DNA polymerases, T4+, T7+, KF+
and Vent, were tested and similar off switches were observed when
the polymerases were combined with 3´ phosphoro-thioate-modified
primers. Based on the new model of this proofreading mechanism (Figure 1),
polymerases with 3´ exonuclease function should have a higher
base discrimination ability over exo-polymerases regardless of the
properties of the substrates used. In addition to comparing nine
different DNA polymerases, Di Giusto et al[6,7,14]
evaluated the effect of dNTP(2´-deoxynucleotide-5´-triphosphate),
ddNTP (2´,3´-dideoxynucleotide-5´-triphosphate),
and acyNTP (acyclo-2´-deoxynucleotide-5´-triphosphate)
on the accuracy of primer extension. The maintenance of high fidelity
with ddNTP and acyNTP allows the Exo+ polymerases to
be applied in both exponential and linear primer extensions in SNP
analysis. The latter was tested using MALDI-TOF MS (Matrix Assisted
Laser Desorption/Ionization Time of Flight Mass Spectrometry) and
is compatible with many other detection formats.
SNP-triggered reversed on/off switch or off/on switch The
SNP triggered reversed on/off switch works in a way complementary
to proofreading 3´ exonuclease-resistant or 3´ labeled
primers. With the introduction of inert allele-specific primers
or proprimers, matched amplicons turn off and mismatched amplicons
turn on DNA polymerization. Inert primers of a perfectly matched
amplicon are not processed by the 3´Exo domain of the high-fidelity
DNA polymerases. In this circumstance, inert primers remain inactive
and no DNA polymerization occurs. In contrast, the inert primers
of mismatched amplicons trigger the 3´exonuclease excision
process by which the mismatched 3´ terminal is removed and
subsequent processes activate the 3´ hydroxyl group for DNA
polymerization. One of the benefits of the reversed on/off switch
is that positive results can screen all three mismatched nucleotides
other than the complementary one, and this is a powerful tool in
mutation detection.
To date two types of 3´ dehydroxylated primers, 3´ phosphorylated
and 3´ hydrogenized, have been evaluated for use in the SNP-triggered
reversed on/off switch. The 3´ phosphorylated primers can always
be extended regardless of whether low-fidelity or high-fidelity
polymerases are used. Fortunately, the 3´ hydrogenized primer
works well as an inert primer that can not be extended without activation
through removal of the 3´ terminal nucleotide residue. The
SNP-operated reversed on/off switch is not a simple candidate method
in mutation detection. One particularly important feature of the
previously developed on/off switch and this new reversed on/off
switch is the identical reaction conditions of the two types of
Exo+ DNA polymerases mediated by the primer extension.
In large-scale SNP scanning, the application of two complementary
assays within one platform, such as multi-well plates or microarrays,
will help to minimize wrongly genotyped SNP sites resulting from
special local sequence contents. With the application of the reversed
on/off switch using the inert primers and the on/off switch using
the 3´ exonuclease-resistant primers, their complementary effect
will help to increase assay sensitivity and reliability in genetic
analysis. The on/off switch provides assays for precise detection
of the location and type of mutation, whereas the reversed on/off
switch serves as a very powerful and efficient assay in unknown
mutation scanning[11]. The reversed on/off switch was
first described in 1998 as a proofreading PCR by Bi and Stambrook[15].
Genomapping applications other than SNP assay
The high-fidelity features of the Exo+ polymerases mediated
on/off and off/on switch can be widely used in a variety of genetic
analysis, other than SNP assays. These include both genotyping and
gene expression profiling. Genotyping focuses on the sequencing
context and gene expression profiling evaluates both the sequence
context and the functional levels of any specific isoforms of a
given gene product. The Exo+ polymerases mediated assays
provide methods useful for these two types of genetic assays simultaneously,
an advantage favor to pharmacogenetic studies. To simplify the terminology,
we refer the combination of genotyping and gene expression profiling
as genomapping analysis.
Analysis of mutations with short deletion/inser-tion Most
of the currently available SNP assays, such as the single base extension
assay, can not discriminate between wild-type alleles and mutants
with small insertions or deletions. The aforementioned data illustrates
the powerful mutation detection ability of the novel on/off switch,
indicating its potential in the analysis of both SNP and other types
of mutations. Except for the deletion or insertion of identical
short repeats, the application of this on/off switch provides an
efficient and high throughput compatible assay for mutation analysis[4-11,16].
Detection of rare alleles using the novel on/off switch
The novel SNP-operated on/off switch is more sensitive than many
conventional mutation analysis methods, including preferential amplification
of the mutant allele, preferential destruction of the wild-type
allele and spatial separation of mutant from wild-type alleles,
by virtue of its use of Exo+ high-fidelity DNA polymerases.
This breakthrough observation of an on/off switch action was repeatedly
confirmed using either short artificial amplicons or natural genomic
DNA templates[11]. Another outstanding feature of the
novel on/off switch is its flexibility and its ability to apply
double switches in a single reaction (for example both the forward
and the reverse primers are 3´-phosphorothioate modified and
allele specific). The double-switch approach does not compromise
sensitivity as is the case in most other primer-extension-based
SNP assays.
Allele frequency estimation in pooled DNA samples The application
of pooled DNA samples in initial studies can help to identify significant
divergence in allele frequencies between case and control populations
for further, more extensive association studies or for haplotype
analysis. Invader technology, Bi-PAP (double directions-pyrophos-phorolysis
activated polymerization), and the SNP-operated on/off switch can
all be used in allele frequency estimation. Among these three assays,
the on/off switch has the advantage of simplicity, sensitivity,
and accuracy. The application of the novel on/off switch in allele
frequency estimation is expected to lower the cost and increase
the accuracy of allele estimation using pooled DNA samples. Because
the analysis of pooled samples requires only a handful of reactions
per SNP, low assay development cost becomes of paramount importance.
High-fidelity gene expression profiling with the novel on/off
switch
In our SNP and mutation detection assays, we have shown that the
on/off switch can recognize a single mismatch in six nucleotides
upstream of the 3´terminus. In most circumstances, Exo+
DNA polymerases can discriminate single mismatched nucleotides several
bases upstream of the primer-3´-termini, but this varies according
to the amplicons and the enzymes[3].
In the early stage of gene expression analysis, mRNA is the target
material. Such methods include Northern blotting and ribonuclease
protection assays. Although these methods could be compatible with
high throughput scalability, the ribonuclease protection assay is
still, to date, the most reliable method in gene expression profiling.
Microarray technology has solved the high throughput issue for genome-wide
gene expression profiling. However, cDNA is an indirect target because
it cannot completely represent the type and abundance of mRNA. False
positives are particularly high when post reverse transcription
amplification by Taq polymerases is used. To minimize the
errors introduced by Taq polymerase-mediated amplification,
high-fidelity DNA polymerases have recently been used in genome-wide
gene expression profiling. Because proofreading polymerases display
up to two orders of magnitude better amplification fidelity, the
application of high-fidelity polymerases has great potential in
improving the reliability of microarray-based gene expression profiling.
In general, false positives in gene expression profiling arise
from three sources: mismatched incorporation in reverse transcription,
mismatched priming, and mismatched incorporation during primer extension.
Although the high-fidelity gene expression assay described in the
present study has nothing to do with the errors introduced from
reverse transcription, it eliminated the mismatchedpriming error
and greatly minimized the mismatched incorporation during primer
extension. Increased fidelity in gene expression profiling not only
benefits assay sensitivity and reliability, it also provides new
applications in genetic analysis. The differential amplification
of the on/off switch allows for the profiling of very rare abundant
transcripts in normal tissues or cancer tissues. It also offers
simple and convenient methods for comparing isoforms, inter-species
or intra-species.
Suggested strategies for somatic mutation load assay
It was almost impossible to analyze rare mutations or mutation
loads before the development of highly sensitive genotyping methods.
The false positives recorded in conventional assay methods, in both
hybridization-based and enzyme-based assays, are higher than the
mutation rate. Theoretically, our recently developed Exo+
polymerase-mediated on/off switch is a good method for examining
rare mutations because it almost eliminates false positives. However,
assay sensitivity is another obstacle facing the detection of rare
mutations. For example, for single-copy genes, 1 mg genomic
DNA only contains 3×105 molecules of relevant genes.
Theoretically, to detect a single mutant out of 1×109-1×1010
wild-type molecules (based on the mutation rate in human somatic
cells per generation), at least mg scale genomic DNA is needed.
These results in two immediate problems: technical difficulties
in obtaining the large amount of human genomic DNA required, and
the inhibitory effect of large amounts of DNA on PCR reactions.
An alternative approach in somatic mutation load analysis is to
use genetic elements with high-copy numbers in the genome. As spontaneous
mutation rates vary among genes and tissues, interspersed repeats
might be a better choice than tandem repeats. A combination of highly
sensitive SNP genotyping methods and a high-copy number of interspersed
repeats may provide a practical and useful strategy for somatic
mutation load assays at the genome level.
Selection of interspersed repeats for somatic mutation load
analysis Short, interspersed nucleotide element (SINE)
repeats, such as Alu elements with a high incidence of CpG dinucleotides
in new Alu inserts, predisposes a higher mutation rate than long,
interspersed nucleotide elements (LINE). Approximately half of the
SNP in young Alu elements fall in the region of CpG dinucleotides.
For somatic mutation load analysis, it appears that the targeting
nucleotides to be chosen need to be carefully sorted. If too many
targets are CpG dinucleotides, the overall estimated somatic mutation
load might be biased and higher than the actual load.
In the case of this example, 1 ng of genomic DNA used in a
somatic mutation load assay with a high-copy number repeat sequence
will have the same power as approximately 1 mg of genomic DNA
when targeting single-copy genes. Another issue regarding our strategies
for somatic mutation load analysis is validation using genomic DNA
samples that have different somatic mutations as expected, such
as DNA from cancer tissue versus DNA from normal tissue, or DNA
from cells in their early passages versus that from late passages.
Alternative opportunities from mitochondrial DNA The
large number of mitochondria present in every mammalian cell (1000-10 000)
makes this extra-nuclear genetic material very useful in somatic
mutation analyses. Furthermore, studies have reported that mitochondrial
DNA (mtDNA) has a mutation rate of one base mutation per 1500-3000
years of evolution, which is believed to be a higher rate than chromosomal
DNA. The existence of homoplasmy and heteroplasmy in mitochondria
mutation and their close relationship with the development of disease
strongly suggests the value of testing the mutation load of mtDNA
in future studies of personalized medicine. Some mtDNA somatic mutation
showed a dose-dependent pattern in causing diseases[17,18]
One technical advantage of sequence comparisons is the very-low
copy numbers of well-matched regions between mitochondrial and chromosomal
DNA. This is especially true for mitochondrial tRNA genes, which
are never observed in more than five copies in chromosomal DNA.
This feature allows somatic mutation load assays to be separately
designed for chromosomal and mitochondrial DNA without preparing
separate DNA samples.
Target simplification by using in vitro amplified DNA
Genomic DNA can be easily simplified and amplified using routine
PCR technology. From a technical point of view, a combination of
the PCR amplification of target DNA and a subsequent mutation load
detection using one of the mutation quantitative analysis methods
outlined can be viewed as a type of nested PCR. An advantage of
this nested strategy is that any chromosomal region can be used
as the target in somatic mutation load analysis.
In addition, many types of RNA are transcribed at highly abundant
levels. Therefore, cDNA obtained immediately from in vitro
reverse transcription or with a low-cycle-number PCR amplification
can be a useful target in somatic mutation load analysis. A potential
drawback of using RNA as the target in somatic mutation load analyses
is that most mutated RNA are not as stable as their wild-type partners,
which may decrease the opportunity for accurately detecting mutations.
Therefore, RNA can act as one of the targets, but can not be used
as the sole target in practical somatic mutation load analyses when
a global approximate of the mutation load is required.
Conclusion
Significant advances after the completion of the Human Genome Project,
including bioinformatic algorithms and highly sensitive SNP genotyping
technologies, have provided great possibilities for studies relating
genotype with phenotype. High-fidelity DNA polymerases have been
approved for use as new and very attractive members among a variety
of enzymes in genetic analysis. The three types of new methods mediated
by proofreading polymerases provide sensitive and reliable tools
for genomapping analyses consisting of genotyping, gene expression
profiling, and mutation load assay. Genotyping, including SNP screening,
is now widely used in pharmacogenetic studies. The most immediate
tasks in somatic mutation load analysis are the selection of the
genetic sequence set as representative targets for mutation assay,
and the validation of these representative sequences using one or
more of the three mutation analysis methods discussed. It is reasonable
to expect that somatic mutation load analysis, especially the mitochondrial
somatic mutation load, will have an important impact on both basic
research and clinical practice.
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