The Advances of DHPLC in SNP and Mutation Detection
Jim Zhu, Transgenomic Inc, Omaha, NE USA
As we are now entering the “post-genomic” era, there are a number of new research
trends. One trend will interrogate the influence of common genetic variants within the
human genome. These common variants, which are often referred to as polymorphisms,
have already been proven to play a role in disease predisposition and progression, drug
toxicity, and general pharmaceutical efficacy. Genetic polymorphisms (particularly
single-nucleotide polymorphisms (SNPs), which account for ~90% of common genetic
variants) are currently used within various association studies for disease research.
Another trend will be an increase in the number of disease researchers directing their
efforts upon focused regions within the genome. These efforts will discover and screen
for various genetic mutations within a functional pathway and reveal their linkages to the
diseases.
The third trend is to provide clinical genetic diagnosis by utilizing the available SNP and
mutation databases for earlier disease prevention and prenatal care. In this application,
accuracy, speed, automation, reliability, affordability, and flexibility, as well as the
ability to detect both known and unknown mutations will be of great importance.
These three trends alone demonstrate the post-genomic era’s requirements for genetic
analysis technologies that can provide high degrees of sample throughput without
sacrificing sensitivity, as well as high levels of automation without sacrificing flexibility.
To address these needs, Transgenomic has developed Wave DHPLC nucleic acid analysis
technology. The Wave DHPLC meets all of the requirements set above for
comprehensive genetic mutation and polymorphism discovery and screening. The
success of the Wave DHPLC approach to genetic analyses is demonstrated in its
applications by many dozens of researchers around the world in a variety of contexts and
demonstrated by many commercial clinical diagnostic services offered with this
technology.
This presentation will focus upon Wave DHPLC applications in clinical genetic
diagnostics and discuss the basic requirements in genetic testing and the complexity of
the sample analysis. Two DHPLC methods: Temperature Modulated Heteroduplex
Analysis (TMHA) and Multiplex Primer Extension (MPE), were developed to optimize
the efficiency in genetic diagnosis for analysis of genes with unknown mutations; genes
with hundreds of mutations but no hot spots; and genes with hot-spot mutations. Many
current available testing procedures and protocols will be given as examples, such as
breast cancer, hereditary nonpolyposis colorectal cancer, polycystic kidney disease, betathalassemia, Dementia, Parkinson, Wilson and DMD diseases. Details of primer design,
PCR conditions, DHPLC gradient and temperature conditions and reference
chromatograms of wildtype and mutants will be presented.
Another focus of this talk will be on the rapid detection of genetic variations in
mitochondria DNA using DHPLC. Mitochondrial DNA is a circular molecule that has
16,569 pb with 37 genes encoding oxidative phosphorylating subunits, two rRNA genes
and 22 tRNA genes. It is present in large numbers in every cell in the body. There are a
number of diseases, such as myopathies, neurological disorders and cancers that have
been associated with the mitochondrial mutations. The ability to detect these mutations
offers significant diagnostic benefits to patients and understanding of these genetic
diseases. Mitochondria DNA is also unique in that it is only inherited maternally, and
has very high copy numbers per cell. It is frequently used in forensic analysis for human
identification where nuclear-based analyses have failed.
Many investigators use direct sequencing to study mitochondria DNA. However, most
pathogenic mitochondrial DNAs mutations are heteroplasmic. Individuals can have more
than one species of mtDNA in varying concentrations in different body fluids or tissues.
Many mtDNA disorders show a wide spectrum of clinical manifestations and variations
in onset, course and progression of the diseases, with different percentages of mutant
mtDNAs. The detection limits of sequencing for heteroplasmic variants are only at 20%
- 30%. Better sensitive detection methods are in demand.
Denaturing high performance liquid chromatography technique is an ideal technique for
identifying heteroplasmic mtDNA mutations due to its superior sensitivity, and can detect
both known and unknown mutations. In conjunction with the Transgenomic MitoScreen
Assay Kit, it offers the ability to scan for the genetic variation over the entire length of
the mitochondrial genome. Importantly, this approach eliminates the need to design and
optimize a specific assay for each individual mutation. No other technology we know
offers the combination of sensitivity, accuracy, versatility, and automation in such a cost
effective format for mtDNA detection.
The MitoScreen Assay Kit uses human genomic DNA as starting material for PCR
amplification of the human mitochondrial genome using a total of 19 primer sets. The 19
amplified fragments are overlapped and ranged in size from 300 – 1500 bp. Four of these
fragments are in the size range 300-550 and are directly used for DHPLC analysis. The
remaining 15 fragments undergo a restriction digestion step (Fig. 1) to produce a
collection of fragments that subsequently analyzed by multiplex DHPLC. An example is
given by analysis of PCR fragment MT10 in different pooled DNA samples at 55oC. The
sensitivity and specificity of DHPLC technique were reported by the analysis of PCR
fragments containing the known A3243G and A8344G mutations. The A3243G mutation
was detected in the samples with heteroplasmy levels of 4.5% and the A8344G mutation
was detected in the samples with heteroplasmy as low as 0.5%.
Forensic application was demonstrated by Dr. Greg LaBeerge, Denver Police
Department, Crime Laboratory Bureau, in his publication on the Forensic Science, 44(3),
281-288, 2003 “Forensic Utility of Mitochondrial DNA Analysis Based on Denaturing
High-Performance Liquid Chromatography”. MtDNA hypervariable regions (HV) 1 and
2 from the mtDNA D-loop were analyzed. They reported 144 pairwise comparison HV1
and HV2 mtDNA fragments were successfully resolved by DHPLC method. One
questioned sample prepared by the test providers was successfully identified as a body
fluid mixture. They predict DHPLC will soon become a standard method in forensic
analysis to resolve mtDNA mixture. Details of the method will be presented.